Fluorescent determination of chloride in nanoliter samples

• Published in: Kidney international Vol. 55; no. 1; pp. 321 - 325
• Main Authors: García, Néstor H., Plato, Craig F., Garvin, Jeffrey L.
• Format: Journal Article Conference Proceeding
• Language: English
• Published: New York, NY Elsevier Inc 01-01-1999; Nature Publishing
• Subjects: Animals; Biological and medical sciences; bromide; Chlorides - analysis; cotransport; DIDS; Fluorescent Dyes; furosemide; In Vitro Techniques; Investigative techniques, diagnostic techniques (general aspects); kidney; Kidney Tubules - metabolism; Medical sciences; Microchemistry - instrumentation; Microchemistry - methods; Microchemistry - statistics & numerical data; Miscellaneous. Technology; Pathology. Cytology. Biochemistry. Spectrometry. Miscellaneous investigative techniques; Perfusion; Quinolinium Compounds; Sensitivity and Specificity; Spectrometry, Fluorescence - instrumentation; Spectrometry, Fluorescence - methods; Spectrometry, Fluorescence - statistics & numerical data; thick ascending limb; furosemide; cotransport; kidney; thick ascending limb; DIDS; bromide; Human; Sensitivity; Biochemical analysis; Fluorescent tracer; Measurement technique; Chlorine ion; Aqueous solution; Sampling; Quantitative analysis
• ISSN: 0085-2538; 1523-1755
• DOI: 10.1046/j.1523-1755.1999.00239.x

Fluorescent determination of chloride (Cl-) in nanoliter samples.
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.
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.
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 mM 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.
We developed an easy, accurate, and sensitive method to measure Cl- concentration in small aqueous solution samples.