Development of a multiple-bile-ion-sensing membrane electrode Sudeshna M. Chatterjea ⇑ , Koustubh Panda ⇑ Department of Biotechnology and Dr. B.C. Guha Centre for Genetic Engineering and Biotechnology, University of Calcutta, Kolkata 700019, West Bengal, India article info Article history: Received 12 April 2013 Received in revised form 21 June 2013 Accepted 28 June 2013 Available online 16 July 2013 Keywords: Multiple-bile-ion-sensing electrode Changing electrode selectivity Electrode characterization Application in kinetics abstract A multiple-bile-ion-sensing polyvinyl chloride-based membrane electrode capable of monitoring any of the three common bile ions in humans, namely, cholate, deoxycholate, and chenodeoxycholate, was developed and characterized. Compared to single-bile-ion-sensing electrodes, it showed a sub-Nernstian response. All other electrode properties were, however, similar, making this a successful replacement for three individual electrodes. With appropriate conditioning, this electrode could repeatedly change selec- tivity without losing membrane activity. It was reproducible, was stable for 5 months, had low response time, and could be used to measure critical micelle concentrations. The lower limit of detection was 10 nM. Selectivity coefficients for various anions with respect to bile ions more or less followed the Hoffmeister series. Plots of R ((Nernst equivalent of slope in the presence of primary ion and a fixed amount of interfering ion)/(slope in the presence of only the primary ion)) vs square root of ionic strength for an interfering ion were linear. One major application of this electrode is its use in kinetics. We have tested its ability to monitor continuously changing bile ion concentrations during their interactions with a biocompatible polymer, polyethylene glycol (6000), and determined rate constants. Ó 2013 Elsevier Inc. All rights reserved. Bile acids constitute a large family of molecules comprising a steroid structure with four rings containing hydroxyl groups pres- ent in different orientations, and a 5- to 8-carbon side chain termi- nating in a carboxylic acid, making them weakly surface active [1,2]. Though these biological detergents are mainly involved in dietary lipid absorption and cholesterol homeostasis, over the past few years they have been shown to also act as signaling molecules [3–7]. To elucidate physiological roles of bile salts in molecular level and to provide a model for lipid protein interaction it is essen- tial to study their interactions with proteins kinetically and at equilibrium. A number of methods are currently available for determining bile salt concentrations in solution, using which bile salt protein interactions have been studied at equilibrium [8]. Reports on kinet- ics of these interactions, however, are scarce because of the lack of a suitable technique for estimating continuously changing bile salt concentrations in solution. This is mainly because surfactants do not have concentration-dependent sharply changing properties ex- cept for surface tension and conductance, which are not suitable for kinetic purposes especially in the presence of salts. To over- come this limitation, we have focused on the use of conventional plastic ion-selective electrodes, a methodology well established in literature that has been used for estimating synthetic surfactants in solution [9–13]. Maulik et al. used a CTAB (cetyltrimethyl ammonium bromide)- selective membrane electrode to study the kinetics of its interac- tions with biopolymers [10–12]. Bile salt-selective membrane elec- trodes could similarly be used for determining kinetic parameters of their interactions with proteins. Some papers in the past have reported preparation of bile salt-selective liquid membrane elec- trodes [13–16] and used them to study their interactions with bo- vine serum albumin at equilibrium [15]. Since it would be a more efficient use of electrodes if a single electrode could be used to monitor multiple ions in solution (selec- tive to a single ion at a time), we have attempted to develop and characterize a polyvinyl chloride (PVC)-based multiple-bile-ion- sensing electrode. Ideally this new electrode could be used to study the interactions of three major bile salts (cholate, deoxycholate, and chenodeoxycholate) with proteins. To establish that this multiple-ion-sensing electrode could truly be a replacement for three single-ion-sensing electrodes, a comparative study with a cholate-selective, a deoxycholate-selective, and a chenodeoxycho- late-selective electrode was done at each step of characterization. Electrode characterization was done in terms of response time, detection limit, drift, stability, lifetime, reproducibility, pH, temperature, and response in comparison to ideal Nernstian behavior. Interfering effects of other bile salts, synthetic surfactants, cholesterol, and inorganic ions during the monitoring of the principal bile ion were studied and selectivity coefficients calculated. In addition to estimating the free bile ion concentration in solution, including physiological fluids, the electrode’s ability to monitor continuously changing bile ion concentration was tested 0003-2697/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ab.2013.06.022 ⇑ Corresponding authors. Fax: +91 033 2461 4849. E-mail addresses: sudeshnachatterjea@hotmail.com (S.M. Chatterjea), panda- k66@yahoo.co.uk (K. Panda). Analytical Biochemistry 441 (2013) 218–224 Contents lists available at ScienceDirect Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio