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C-SNARF-1 as a Fluorescent Probe for pH Measurements in Living Cells: Two-Wavelength-Ratio Method versus Whole-Spectral-Resolution Method

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A-C. Ribou , J. Vigo and J-M. Salmon

Laboratory of Physico-Chemical Biology of Integrated Systems, University of Perpignan, F-66860, Perpignan, France

J. Chem. Educ., 2002, 79 (12), p 1471

DOI: 10.1021/ed079p1471

Publication Date (Web): December 1, 2002

Section:

History, Education, and Documentation

Abstract

Measuring intracellular concentrations of ions (H⁺, Ca²⁺, Mg²⁺) is one of the challenges of modern cell biology. Fluorescence techniques can be used for this purpose since they are noninvasive, specific, and in general sensitive enough to require only low concentrations of the probes. In this paper, we describe two methods to measure pH by fluorescence spectroscopy and a way to extend the measurement to living cells. We use the fluorescent probe, C-SNARF-1, which exhibits a spectral shift of 45 nm upon proton binding. The two-wavelength-ratio method is applied to the determination of an unknown pH in aqueous solution. The whole-spectrum-resolution method is used for the same solution for comparison and then applied on a prerecorded spectrum of a suspension of living cells. The methods described here have been adapted for laboratory classes of undergraduate students. The lab employs experimental fluorescence techniques and computational data analysis that account for the fluorescence of cellular flavoproteins. The pedagogic purpose of the experimental design is to introduce students to the concepts, equations, and validity of both methods. In addition, skills in record keeping and data analysis using Microsoft Excel Solver are developed.

Keywords (Audience):

Upper-Division Undergraduate

Keywords (Domain):

Analytical Chemistry

Keywords (Pedagogy):

Hands-On Learning / Manipulatives

Keywords (Subject):

Acids / Bases

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This article has been cited by 1 ACS Journal articles (1 most recent appear below).

Spectrophotometric Determination of the Dissociation Constant of an Acid-Base Indicator Using a Mathematical Deconvolution Technique
Krystyn P. Alter , John L. Molloy and Emily D. Niemeyer
Journal of Chemical Education2005 82 (11), 1682
- Spectrophotometric Determination of the Dissociation Constant of an Acid-Base Indicator Using a Mathematical Deconvolution Technique
  Krystyn P. Alter , John L. Molloy and Emily D. Niemeyer
  Journal of Chemical Education2005 82 (11), 1682
  A laboratory experiment that uses a spectrophotometric method to determine the apparent acid-dissociation constant (Ka') of neutral red, a common acid–base indicator, has been developed. The experiment incorporates a unique data analysis technique using a ...
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