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A new sensor is described based on a phosphorescent metalloporphyrin dye incorporated in a polymeric membrane, which allows measurement of the two important analytes, oxygen and pH. In such a sensor, the bifunctional dye is quenched by O(2) altering its phosphorescence intensity and lifetime (0-21 kPa O(2)), whereas protonation of the dye causes a major change in the absorption spectrum and also reduces the phosphorescence intensity. As a result, quantification and continuous monitoring of the two analytes can be achieved by (i) simultaneous phosphorescence intensity (O(2), pH) and lifetime (O(2)) measurements and (ii) parallel phosphorescence lifetime (O(2)) and ratiometric absorbance (pH) measurements. Although the first method generally requires sensor calibration in intensity mode, the second provides internal referencing and calibration-free capabilities for both analytes. This approach can be extended to sensing of some other analytes.

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A simple, miniaturized, and automated screening assay for the determination of total aerobic viable counts in fish samples is presented here. Fish tissue homogenates were prepared in peptone buffered water medium, according to standard method, and aliquots were dispensed into wells of a 96-well plate with the phosphorescent, oxygen-sensing probe GreenLight. Sample wells were covered with mineral oil (barrier for ambient oxygen), and the plate was monitored on a standard fluorescent reader at 30°C. The samples produced characteristic profiles, with a sharp increase in fluorescence above the baseline level at a certain threshold time, which could be correlated with initial microbial load. Five different fish species were analyzed: salmon, cod, plaice, mackerel, and whiting. Using a conventional agar plating method, the relationship between the threshold time and total aerobic viable counts load (in CFU per gram) was established, calibration curve generated, and the test was validated with 169 unknown fish samples. It showed a dynamic range of 10(4) to 10(7) CFU/g, accuracy of ± 1 log(CFU/g), assay time of 2 to 12 h (depending on the level of contamination), ruggedness with respect to the key assay parameters, simplicity (three pipetting steps, no serial dilutions), real-time data output, high sample throughput, and automation. With this test, quality of fish samples, CFU-per-gram levels, and their respective time profiles were determined.

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