Daniel Ammann

Ion-Selective Microelectrodes Principles, Design and Application

With 153 Figures and 42 Tables

Springer-Verlag Berlin Heidelberg GmbH

PO Dr. Daniel Ammann

Laboratorium fUr Organische Chemie ETH-Zentrum, Universitatstra13e 16 CH-8092 Zurich

ISBN 978-3-540-16222-3

Library of Congress Cataloging-in-Publication Data. Ammann, Daniel, 1947-lon-selective microelectrodes. Bibliography: p. Includes index.!. Electrodes, Ion selective. LTitle. QD571.A48 1986 541.3'724 85-30423

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© Springer-Verlag Berlin Heidelberg 1986 Originally published by Springer-Verlag Berlin Heidelberg New York Tokyo in 1986

The use of registered names, trademarks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use.

The publisher cannot assume any legal responsibility for given data, especially as far as di­rections for the use and the handling of chemicals are concerned. This information can be obtained from the instructions on safe laboratory practice and from the manufacturers of chemicals and laboratory equipment.

2152-3140-543210

ISBN 978-3-540-16222-3 ISBN 978-3-642-52507-0 (eBook)DOI 10.1007/978-3-642-52507-0

For Madeleine, Desiree, Fabienne, Noemie

Preface

The microelectrode technique is today the most widely used method in electrophy­siology. Microelectrodes offer a unique approach to measurements of electrical pa­rameters and ion activities of single cells. Several important breakthroughs in trans­port physiology have arisen from microelectrode studies. Undoubtedly, there is a progressively wide-spread use of conventional and ion-selective microelectrodes.

Due to their particular dimension and properties micro electrodes are exclusive­ly applied to measurements on living matter. This must have many consequences to my thoughts on experiments with microelectrodes. In this book, my concern is fo­cussing on the description of an intracellular method that should lead to reliable in­formation on cellular parameters. The methodical basis for any meaningful applica­tion is treated extensively. However, technical perfection and accurate results are not the only concern when working on animals and human beings. Rather, my thoughts are governed by the intellectual and moral mastery of the experimental ap­proach on living subjects.

A measurement with microelectrodes usually necessitates the sacrifice of an ani­mal. This is an immense fact, and means that the knowledge gained by the experi­ment must justify the death of a living subject.

Through the availability of a technology as described here, ethics has to argue with scientific developments which have a causal reach into the future. It is not utopic to expect after the mastery of matter (nuclei of elements) and of heredity (recombinant DNA) a third scientific revolution: the intervention on human be­haviour by the brain neurosciences. These extraordinary scientific outcomes in­clude both the beautiful knowledge on life and the risk of going ecologically or an­thropologically wrong.

I am aware to describe only one distinct approach to the reality of a living system out of many others. I agree with the striking example given by P. Feyerabend (Wissenschaft als Kunst, Suhrkamp, Frankfurt, 1984, p. 17 ff.) of the experiment of Brunelleschi, the narrow-minded interpretation of the experiment by Alberti and the subsequent beautiful criticism of Leonardo da Vinci. Brunelleschi's experiment was the following: he paints from the entrance of Santa Maria del Fiore at Firenze with great exactness and beauty the front of San Giovanni at the opposite side of the piazza. While painting with his back turned to the piazza he is looking into a polished mirror where the facade of San Giovanni and the sky is reflected. After­wards, he makes a small hole in the middle of the realistic painting. The observer (the experimentator) standing in the door of Santa Maria del Fiore, exactly where the painting has been made, holds in one hand a mirror and is guiding with the other hand the painting to his eye. Now, he sees in the mirror the painted facade of

VIII Preface

San Giovanni and the moving clouds at the sky. The mirror is removed, and, the view does not change although he views reality now. Undoubtedly, the painting-ex­periment of Brunelleschi has the character of a scientific approach since it tries to describe reality under very controlled experimental conditions. Based on this exper­iment Alberti erects a doctrine for painting. He reduces painting to a geometrical problem. Leonardo da Vinci, however, recognizes that such a theory is valid only under very restricted conditions. The perception of man is much more versatile than it can be described by one distinct approach.

Indeed, an intracellular experiment with microelectrodes is a similar fascinating event like Brunelleschi's painting. Its description of reality is however limited too. Thus, when I am performing microelectrode studies on animal cells I do not look at the living subject itself but at the electrical signals generated at the microelectrode which is connected to a complex electronic equipment. It is exclusively the resulting pen recording that allows me to interpret observations on the living matter. The real cell is hidden behind the impressive, however limited, information of the measured potentials. Accordingly, the result of the experiment is one specific scientific view of the cell and is only valuable for the conditions imposed. Although the results are describing life from the physiological point of view they overlook in many respects other aspects of the life of the cell, of the sacrificed animal, and of the environment it came from. Physiology and cell biology never offer a complete message of living structure. In other words, the cells and organisms have several aspects, the descrip­tion given in this book is satisfying the current scientific approach only. Reality is a relative term, and a reduction to a single description appears to me a risk and a loss.

Preface IX

I am very grateful to Dr. Peter Anker, Dr. Daniel Erne, Dr. Urs Oesch, Dr. Erna Pretsch and Prof. W. Simon for their comments and advices on the manuscript. Fur­ther I wish to thank Madeleine Ammann, Dr. Alan Berry, Catriona Tedford and Dr. Dorothee Wegmann for their most valuable help in correcting the manuscript. I also thank Brigitte Marti for the typing of the manuscript. Finally, it was important for me to visit the physiological laboratories of Dr. Annen Kurkdjian at Gif-sur-Yvette and of Prof. Florian Lang at Innsbruck. I am grateful for the discussions at these places.

Daniel Ammann Zurich. November 1985

Table of Contents

1 Introduction. . . . . . . . . . . . . . . .

2 Classification of Ion-Selective Electrodes

2.1 Types of Membranes . . 2.2 Electrode Arrangements 2.3 Concluding Remarks . .

3 Natural and Synthetic Neutral Carriers for Membrane Electrodes .

3.1 Carriers as a Class of Ionophores .............. .

3.2 Molecular Aspects of the Ion-Selectivity of Neutral Carriers 3.2.1 General Remarks ............... . 3.2.2 Models for the Design of Neutral Carriers ..

3.2.2.1 Corey-Pauling-Koltun (CPK) Models 3.2.2.2 Electrostatic Models ......... . 3.2.2.3 Quantum Chemical Model Calculations.

3.2.3 Selectivity-Determining Parameters. 3.2.3.1 General Remarks ........ . 3.2.3.2 Binding Sites ........... . 3.2.3.3 Coordination Number and Cavity 3.2.3.4 Arrangement of the Binding Sites 3.2.3.5 Size of the Carrier ........ .

3.2.4 Properties of Neutral Carriers for Analytically Relevant Membrane Electrodes . . . . . . . . . . . . 3.2.4.1 Permselectivity. . . . . . . . . . . . 3.2.4.2 Ion- and Ligand-Exchange Kinetics 3.2.4.3 Lipophilicity ............ .

. . . . .

3.3 Highly Cation-Selective Neutral Carriers for Liquid Membrane

1

3

3 5 7

9

9

13 13 13 14 14 15 18 18 18 19 19 21

21 23 26 28

Electrodes . . . . . . . . . . . . . . . . . . . . 30

3.4 Synthesis of Cation-Selective Neutral Carriers 33 3.5 Carriers for Anions . 33 3.6 Concluding Remarks . . . . . . . . . . . . . . 40

XII Table of Contents

4 Liquid Membrane Electrodes Based on Neutral Carriers 43

4.1 Parameters Determining Basic Properties of Solvent Polymeric Membranes . . . . . . . . . . . 45 4.1.1 Membrane Composition. 45 4.1.2 Membrane Solvent . . 47 4.1.3 Membrane Matrix . . . . 50 4.1.4 Membrane Additives . . . 53

4.2 Ion-Selective Membrane Solutions for Microelectrodes 54 4.2.1 Composition of Membrane Solutions . 54 4.2.2 Solvents for Membrane Solutions . 55 4.2.3 Additives for Membrane Solutions 56

4.3 Concluding Remarks. . . . . . . . . . . 61

5 Potentiometric Measurements of Ion Activities with Neutral Carrier-Based Electrodes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

5.1 Cell Assemblies 5.2 The Membrane Potential . . . . . . . . . . . . . . . 5.3 The Nernst and the Nicolsky-Eisenman Equations. 5.4 Electrode Function and Detection Limit .. . . . . 5.5 Selectivity Factors. . . . . . . . . . . . . . . . . . . 5.6 Activity Coefficients. The Debye-Hiickel Formalism. 5.7 Liquid-Junction Potentials. The Henderson Formalism 5.8 Stability and Reproducibility of EMF. 5.9 Response Time .. 5.10 Lifetime ...... . 5.11 Anion Interference . 5.12 Concluding Remarks

6 Construction of Liquid Membrane Microelectrodes .

6.1 Glass Tubings . . . . . . . . . . . . . . . . . . 6.2 Micropipettes . . . . . . . . . . . . . . . . . . . . . . . . . 6.3 Single- Versus Double- or Multi-Barrelled Microelectrodes 6.4 Electrode Bevelling . 6.5 Tip Observation . 6.6 Silanization . . . . . 6.7 Electrode Filling .. 6.8 Electrode Shielding . 6.9 Reference Microelectrodes . 6.10 Concluding Remarks ....

65 67 68 69 70 73 78 81 82 88 92 95

97

97 100 103 106 108 109 113 114 115 117

Table of Contents XIII

7 Use of Neutral Carrier-Based Liquid Membrane Microelectrodes 119

7.1 Concepts of Use. . . . . . . 119 7.2 Electrode Testing . . . . . . 122 7.3 Calibration and Evaluation 123 7.4 Impalement Criteria 126 7.5 Cell Damage ........ 129 7.6 Cell Contamination. . . . . 133 7.7 Tip Potentials and Electrical Shunts 140 7.8 Limitations Due to Selectivity 143 7.9 Concluding Remarks. . . . . . . . 152

8 General Aspects of Intracellular Measurements of Ions. 153

8.1 The Intracellular Space as the Measuring Sample 153 8.2 Ion Activity, Concentration of Free Ions, Total Concentration 167 8.3 In vitro and in vivo Measurements. . . . 169 8.4 Organelles, Single Cells, Cell Population . . . . . . . . . . . . 172 8.5 Cell Compartmentation ..................... 176 8.6 Method Specific Properties of Intracellular Measurements of Ions 188 8.7 Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . 193

9 Neutral Carrier-Based Liquid Membrane Microelectrodes . 195

9.1 Intracellular Measurements of H+ . . . . . . . . . . 195 9.1.1 Distribution of Weak Acids and Weak Bases. 195 9.1.2 Optical Techniques. . . . . . . . . . . . . . . 197 9.1.3 31p Nuclear Magnetic Resonance Spectroscopy 199 9.1.4 Potentiometry .................. 204

9.1.4.1 Survey of H + -Selective Microelectrodes . 204 9.1.4.2 Available Liquid Membrane Microelectrodes . 205 9.1.4.2.1 Microelectrodes Based on 3-Hydroxy-N -octyl-

picolinamide . . . . . . . . . . . . . . . . . . . 205 9.1.4.2.2 Microelectrodes Based on the Na + Salt of the Anti-

biotic Nigericin ................... 205 9.1.4.2.3 Microelectrodes Based on p-Octadecyloxy-m-chloro-

phenyl-hydrazone-mesoxalonitrile . . . . . . . 207 9.1.4.2.4 Microelectrodes Based on Tri-n-dodecylamine 207

9.1.5 Comparison of the Different Methods 213 9.1.6 Concluding Remarks .... 217

9.2 Intracellular Measurements of Li + .... .

9.3 Intracellular Measurements of Na + .... . 9.3.1 Techniques Used in Studies of Intracellular Na +

217

222 222

XIV Table of Contents

9.3.2 Survey of Na + -Selective Microelectrodes ...... . 9.3.3 Available Liquid Membrane Microelectrodes .... .

9.3.3.1 Microelectrodes Based on Potassium Tetrakis (p-chlorophenyl) borate . . . . . . . . . . . .

9.3.3.2 9.3.3.3

9.3.3.4

Microelectrodes Based on the Antibiotic Monensin . Microelectrodes Based on the Neutral Carrier ETH227 .................... . Microelectrodes Based on the Neutral Carrier ETH 157 ..... .

223 224

224 224

226

231 9.3.4 Concluding Remarks .......... 232

9.4 Intracellular Measurements of K + . . . . . . . 233 9.4.1 Survey of K + -Selective Microelectrodes 233 9.4.2 Comparison of Properties of Classical Ion-Exchanger and

Neutral Carrier-Based Microelectrodes 235 9.4.3 Concluding Remarks ................. 240

9.5 Intracellular Measurements ofMg2+ ............ 240 9.5.1 Mg2+ Null Point for Permeabilized Cell Membranes 240 9.5.2 Metallochromic Indicators ........... 242 9.5.3 31p Nuclear Magnetic Resonance Spectroscopy 243 9.5.4 Potentiometry .................. 245

9.5.4.1 Mg2+ -Selective Carriers. . . . . . . . 245 9.5.4.2 Microelectrodes Based on the Neutral Carrier

ETH 1117 .......... . 9.5.5 Comparison of the Different Methods 9.5.6 Concluding Remarks .... .

9.6 Intracellular Measurements ofCa2+ .. .

9.6.1 Photoproteins .......... . 9.6.1.1 Origin of Bioluminescence 9.6.1.2 Availability of Photoproteins 9.6.1.3 Properties and Use of Photoproteins .

247 252 253

253 253 253 254 255

9.6.1.4 Measurement of the Luminescence of Photo proteins 259 9.6.2 Metallochromic Indicators .... 260

9.6.2.1 Currently Used Indicators ............ 260 9.6.2.2 Properties of the Indicators . . . . . . . . . . . . 260 9.6.2.3 Measurements with Metallochromic Indicators. 263

9.6.3 Potentiometry ....................... 265 9.6.3.1 Survey of Ca2 + -Selective Microelectrodes . . . . 265 9.6.3.2 Course of the Development of Optimized Neutral

Carrier-Based Ca2+ Microelectrodes. . . . . . . . 268 9.6.3.3 Properties of Currently Used Microelectrodes Based on

the Neutral Carrier ETH 1001 ........... 273 9.6.3.4 Calibration of Ca2 + -Selective Microelectrodes for

Intracellular Studies . . . . . . . . . . . . . . . . . 275

Table of Contents XV

9.6.4 Comparison of the Different Methods 276 9.6.5 Concluding Remarks 277

9.7 Other Microelectrodes . . . . . . . . 279

10 Impact of Neutral Carrier Microelectrodes

10.1 Applications of Neutral Carrier-Based Microelectrodes 10.2 Animal Experimentation ......... . 10.3 Neurosciences. The Mind-Body Problem.

11 References . .

12 Subject Index

281

281 296 298

305

339