Fluorescent determination of chloride in nanoliter samples

Background. Measurements of Cl- in nanoliter samples, such as those collected during isolated, perfused tubule experiments, have been difficult, somewhat insensitive, and/or require custom-made equipment. We developed a technique using a fluorescent Cl- indicator, 6-methoxy N-(3-sulfopropyl) quinolinium (SPQ), to make these measurements simple and reliable. Methods. This is a simple procedure that relies on the selectivity of the dye and the fact that Cl- quenches its fluorescence. To measure millimolar quantities of Cl- in nanoliter samples, we prepared a solution of 0.25 mM SPQ and loaded it into the reservoir of a continuous-flow ultramicrofluorometer, which can be constructed from commercially available components. Samples were injected with a calibrated pipette via an injection port, and the resultant peak fluorescent deflections were recorded. The deflections represent a decrease in fluorescence caused by the quenching effect of the Cl- injected. Results. The method yielded a linear response with Cl- concentrations from 5 to 200 mM NaCl. The minimum detectable Cl- concentration was approximately 5 mM. The coefficient of variation between 5 and 200 mM was 1.7%. Resolution, defined as two times the standard error divided by the slope, between 10 and 50 mM and between 50 and 200 nM was 1 mM and 2.6 mM, respectively. Furosemide, diisothiocyanostilbene-2,2'-disulfonic acid and other nonchloride anions (HEPES, HCO3, SO4, and PO4) did not interfere with the assay, whereas 150 mM NaBr resulted in a peak height greater than 150 NaCl. In addition, the ability to measure Cl- did not vary with pH within the physiological range. Conclusion. We developed an easy, accurate, and sensitive method to measure Cl- concentration in small aqueous solution samples.