One or more high intensity light emitting diodes (LEDs), after proper bandwidth filtering, are used commercially to illuminate
the sensing patch, which is prepared with a dye that causes reflected light (measured at a different wavelength) to vary in
a well-defined and consistent way with changes in the measured parameter.
As an example, a system for measuring DO can be designed based on the principal that oxygen quenches, in a dynamic and well-defined
manner, the fluorescence of any fluorophore with a lifetime longer than 10 ns. Such a sensor will use a modulated, properly
filtered light source (a high intensity blue LED, for example) to excite a patch treated with a specific compound, commonly
a ruthenium-based or platinum-based fluorophore, although other compounds have been used.
The patch, when excited, will reflect light, which differs from the incident light in wavelength, phase, and intensity. The
amount of phase shift and difference in intensity are dependent on the DO level in the immersion liquid. The reflected light,
properly filtered, can be captured using a common photodetector. Either change in intensity or shift in phase can be used
as the primary measured variable, although intensity is less desirable because of possible errors caused by background noise,
ambient light conditions, and changes in incident intensity caused by aging of the excitation LED and associated electronics.
Although more difficult to measure electronically, phase shift is thus the preferred parameter in this case.
Measurement instruments for pH, CO2, and other parameters can be designed in a similar manner. Despite the differences in optics, chemistry, and methodologies,
these parameters are measured with instruments using physically similar designs and apparatus, and are based on the same principal
of exciting a patch treated with a specific fluorophore with one or more light sources, and measuring and analyzing the reflected
light. A pH sensor, for example, might use a patch treated with a dye such as hydroxypyrene trisulfonic acid (HTPS), which
exhibits two excitation wavelengths that correspond to the acid and its conjugate base. Using a pair of high intensity LEDs
to excite the patch, one ultraviolet and one blue, the ratio of emission intensities can be used to determine pH. CO2 can
be measured in a similar manner. Research is currently being directed toward the robust design of sensors for more exotic
parameters such as alcohols, green fluorescent protein, and glucose.
Some care must be used in the design and fabrication of the sensing patch. Typically, such patches comprise a number of thin
layers: a protective layer made of mylar or similar material to protect the adhesive until the patch is ready to be pressed
in place; the adhesive layer; a support layer to provide some degree of rigidity to the patch; the sensing layer; and a cover
layer that provides optical isolation for the sensing layer. The sensing layer contains the dye, which is usually immobilized
in some fashion to minimize leaching of the dye into the medium. For example, for DO, the dye can be immobilized in an oxygen
permeable polymer film or absorbed on silica gel. The cover layer is required to prevent ambient light from unduly affecting
the sensor's readings.
With minor modifications, fluorescence-based sensors also can be used to measure process parameters in a gas stream as well
as a liquid. By using three such sensors with a bioreactor system (to measure DO in the inlet and outlet gas streams and in
the culture medium), it is easy to determine total oxygen uptake rate over a period of time. If the uptake rate of the cell
line is known, this provides an easy way to determine viable cell density during a culture, and thus the optimal harvesting
Because there is no probe entering the culture vessel with a fluorescence-based sensor, these sensors can be used in vessels
of only a few milliliters in size. This is because the illuminated area of the patch (the only component that must be placed
in the vessel) need be no more than a few millimeters in diameter, or even smaller, if fiber optics are used as a light source
and receiver in place of the excitation LED and photodetector. Of course, the use of fiber optics has its own unique set of
problems, but it has enabled the technology to be used in applications that otherwise have very limited space for the accommodation
of sensors for parametric measurement.
Fluorescence-based technology has a number of advantages over conventional probe-type sensors. The ability to inexpensively
measure process parameters in small vessels allows key operational parameters to be accurately measured much earlier in the
development cycle than would be possible otherwise, such as at the flask or well-plate stage rather than the bioreactor stage.
This permits early determination of optimal media formulation and cell culturing conditions, and thus facilitates scale-up
from glassware to laboratory-scale and production-scale bioreactors.