Time-resolved pH/pO(2) mapping with luminescent hybrid sensors

A method for simultaneous and referenced 2D mapping of pH and pO(2) is described. The experimental setup combines a fast gateable CCD camera as detector, a LED as excitation light source and a single-layer sensor membrane as optical transducer. The planar optode comprises a lipophilic fluorescein derivative (lifetime tau similar to 5 ns) and platinum(II) mesotetrakis(pentafluorophenyl)porphyrin (tau similar to 70 mu s in the absence of a quencher) immobilized in a hydrogel matrix. Depending on the fluorescent pH indicator, a pH transition in the physiological range (pH 6-pH 8) or in the near-basic region (pH 7-pH 9) can be achieved. The measuring scheme involves the time-resolved acquisition of images in three windows during a series of square-shaped excitation pulses. A method allowing the calculation of both parameters from these three images is presented. The pH/pO(2) hybrid sensor incorporating the pH indicator 2',7'-dihexyl-5(6)-N-octadecyl-carboxamidofluorescein was characterized in detail. The pH and pO(2) were determined with a maximum deviation of 0.03 pH unit and 6.5 hPa pO(2), respectively, within the range of pH 7.6-pH 8.7 and 0-200 hPa pO(2) in test measurements. The ionic strength (IS) cross-sensitivity was found to be relatively small (Delta pH/Delta IS < 3.5 x 10(-4) mM(-1) and Delta pO(2)/Delta IS <-0.053 hPa mM(-1) at a transition from 0.5 to 0.1 M IS). Whereas a strong temperature effect on the sensor signal was observed (Delta pH/Delta T = 0.011-0.034 K-1 and Delta pO(2)/Delta T = 1.85-7.17 hPa K-1 in the range from 277 to 308 K). Examples of pH/pO(2) images obtained in natural marine sediment are presented.