A fast and durable ratiometric pH microoptode that is highly accurate, precise, sensitive, reversible, and reproducible over the physiological ranges of pH, ionic strength, and temperature has been developed. The sensing site consists of 5 (and 6)-carboxynaphthofluorescein (CNF) entrapped in a polyacrylamide gel matrix via photopolymerization at the silanized end of an optical fiber with a diameter of 2 (pulled) or 125 microns (unpulled). The optode's precision for the pH 6.3-8.4 range in rat embryos, sera, or physiological (Earle's and Tyrode's) buffers was found to be better than +/- 0.03 pH unit. The pulled and unpulled optodes have respective upper limit response times of 1 and 400 ms for 1-pH-unit change. Over a 7-week period, they retain sensitivity for 600 and 10,000 measurements, respectively. Ratiometric measurements are made using a pH-sensitive emission peak on each side of an isosbestic point. The CNF microoptode is most suitable for biological applications because of its essentially linear response over the pH 7-8 range, its high sensitivity (slope about 2), and its almost perfect correlation with a pH macroelectrode. Furthermore, errors introduced by photobleaching, leaching, quenching, optode movement, and excitation source fluctuations are minimal.
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