Development and evaluation of a fluorescence emission ratio-based fiber opticpH measurement system for use in monitoring changes in tumor pH during clinical hyperthermia

The pH of the tumor microenvironment may be important in assessment of response to hyperthermic therapy. Little clinical in vivo data is available during such therapy due to the inherent limitations of the microelectrode technique in the presence of a microwave field. A fiber optic pH measurement system, due to its dielectric nature, provides a method for overcoming such limitations. Optrodes sensitive to pH have been studied based on absorption and fluorescence. Absorption based optrodes are difficult to fabricate due to the complex nature of the required optical geometry. Fluorescence based optrodes have been developed based upon either a single emission intensity measurement at a specific wavelength or the ratio of two emission intensities, at the same wavelength, following sequential excitation. Single intensity measurements are prone to substantial errors introduced by differences in the ionic strength and temperature of the samples as well as by fluorophore leakage, photobleaching, and fluctuations in the intensity of the excitation source. The ratio technique minimizes the above sources of measurement error, but the need for a sequential excitation results in instrumentation that may be too complex and expensive for routine use. Using the dual emission pH sensitive fluorophore 1,4-dihydroxyphalonitrile (1,4-DHPN), a simple ratio based optical pH measurement system was constructed. Optrodes were fabricated using glass capillary tubes $<$1 mm in diameter with a Cuprophan membrane fixed at one end. The 1,4-DHPN was encapsulated in 4:1 DPPC/DPPG containing LUV in order to limit fluorophore loss and extend the sensor lifetime. A 2 mole percent quantity of gramicidin was added to the lipid phase, during preparation of the LUV, in order to insure rapid equilibration of hydrogen ions across the lipid bilayer. A flashlamp excitation source was used in conjunction with a single optical fiber to excite the fluorophore and to collect its fluorescence. Emission wavelengths of 488 and 434 nm were detected using narrowband interference filters in the optical subsystem. The electronics subsystem was used to electronically process the resultant signals before digitization. Ratios were computed digitally in real time using an Apple 2E microcomputer. This ratio based fiber optic pH measurement was able to to measure pH values in the 6.5-7.5 range with a standard deviation of better than 0.1 pH unit. Over this range a maximum standard deviation of 0.007 pH units/$\sp\circ$C was measured. The time constant of these optrodes was determined to be 3.2 minutes when measured in 305 mOsm phosphate buffer. The time constant in whole blood increased to 10.0 minutes due to a decrease in the hydrogen ion permeability of the LUV membrane. This is most likely due to the blockage of gramicidin channels by divalent cations in the blood plasma.U of I OnlyETDs are only available to UIUC Users without author permissio