Fluorescent determination of chloride in nanoliter samples

Background. Measurements of Cl- in nanoliter samples, such as those collect ed during isolated, perfused tubule experiments, have been difficult, somew hat insensitive, and/or require custom-made equipment. We developed a techn ique using a fluorescent Cl- indicator, 6-methoxy N-(3-sulfopropyl) quinoli nium (SPQ), to make these measurements simple and reliable. Methods. This is a simple procedure that relies on the selectivity of the d ye and the fact that Cl- quenches its fluorescence. To measure millimolar q uantities of Cl- in nanoliter samples, we prepared a solution of 0.25 mM SP Q and loaded it into the reservoir of a continuous-flow ultramicrofluoromet er, which can be constructed from commercially available components. Sample s were injected with a calibrated pipette via an injection port, and the re sultant peak fluorescent deflections were recorded. The deflections represe nt a decrease in fluorescence caused by the quenching effect of the Cl- inj ected. Results. The method yielded a linear response with Cl- concentrations from 5 to 200 mM NaCl. The minimum detectable Cl- concentration was approximatel y 5 mM. The coefficient of variation between 5 and 200 mM was 1.7%. Resolut ion, 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. F urosemide, diisothiocyanostilbene-2,2'-disulfonic acid and other nonchlorid e anions (HEPES, HCO3, SO4, and PO4) did not interfere with the assay, wher eas 150 mM NaBr resulted in a peak height greater than 150 NaCl. In additio n, 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.