Biochimica et Biophysica Acta, 762 (1983) 337-343

Elsevier Biomedical Press

337

BBA 11150

APPLICATION OF ANTIMONY MICROELECTRODES TO INTRACELLULAR pH M O N I T O R I N G

CHRISTIAN GIAUME

and RAYMOND T. KADO b

a C H U Pitie-Salpetriere, Laboratoire de Physiologie, I N S E R M U3, 91 Boulevard de l'H6pital, F-75013 Paris and b Laboratoire de
Neurobiologie Cellulaire, CNRS, Gif - sur- Yvette (France)

(Received January 10th, 1983)

Key words: Antimony microelectrode," Intraeellular p H

Some novel studies of the properties of the antimony microelectrode used for intracellular pH measurements
are described. First, it is shown that currents in the picoampere range, such as those encountered as leakage
in some electrometers, induce important changes in pH sensitivity. The response time of the electrode has
also been measured and indicates that the electrode exhibits a rapid time course which would be very useful
for dynamic cytoplasmic pH investigations. An example of internal pH recording during cellular acidification
in Xenopus laevis oocyte is also presented.

Introduction
Measurements of intracellular pH using microelectrodes have been greatly advanced in the last
10 years, principally through the work of Thomas
[1 ]. The glass pH-microelectrode, particularly with
the recessed tip, has become the standard for
critical intracellular pH measurements. These electrodes are, however, difficult to fabricate, especially if a very fine tip is desired, and difficult to
use owing to a high resistance. In those instances
where only an indication of the intracellular pH at
rest or rapid response to pH shifts are needed, the
glass electrode may seem to require too great an
effort to be worthwhile. In such cases, the antimony (Sb) microelectrode may prove to be a valid,
easily made alternative.
The antimony electrode has had a long history
in pH measurements [2]. Unlike the selective glass
electrode, the Sb electrode depends on a metalmetal oxide to solution interface for its pH sensi-

Abbreviation: Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid.

0167-4889/83/0000-0000/$03.00 1983 Elsevier Science Publishers

tivity. As such, it is intuitively evident that many

factors, ions, organics, gases and currents, may
affect the measured potential which should be due
only to the H + activity at the tip. In spite of these
limitations, the Sb electrode has been used over
the years in many kinds of biological pH studies

[3].
Recently, there has appeared a series of articles
by Fujimoto and coworkers [4-7] in which the
authors have systematically re-evaluated the Sb
microelectrode in relation to (1) the pH-sensitive
glass microelectrode, (2) the physiochemical characteristics and the effects of the buffer used for
calibration, (3) the temperature coefficient and
oxygen effects, and (4) the effects of proteins in
the measured solution. In general, these studies
have shown that under normal conditions the Sb
electrode can provide pH estimated to + 0.2 U or
better against a standard glass microelectrode. Such
a performance is obtained for Sb electrodes having
slopes greater than - 4 5 mV per unit pH and
which did not show drifts greater than 4 mV
before and after calibration in buffers having about
the same ionic strengths as that expected in the
measured medium.

338
We report here the results of some studies relating to the speed of response of these electrodes,
the effects of leakage currents on the p H measurements and some results obtained with intracellular
recordings.
Material and Methods

Microelectrode fabrication
We have used a somewhat simpler procedure
[4,8], for the fabrication of the Sb electrodes:
purified Sb (99.999%) as obtained from most
chemical supply houses was melted in a pyrex
beaker under 1-2 cm of granulated bone charcoal
over an ordinary bunsen flame. The charcoal is
simpler to implement than a nitrogen gas system
to prevent the rapid oxidation of the melted Sb,
and the small quantity of resulting ash does not
appear to affect the performance of the electrodes.
A piece of borosilicate micropipette tubing, 1 m m
inside diameter about 10 cm long, without internal
fiber (Clark Electromedical Instruments CG150T),
was prepared with an appropriate length of flexible tubing slipped over one end. When the Sb was
fluid, the pipette tubing was quickly introduced
into the melt and, with a 10 ml plastic syringe, the
Sb was quickly aspirated. This must be done rather
sharply to be sure to fill a 4 - 5 cm length of the
pipette before the Sb solidifies. Several such tubes
were filled in succession until the molten Sb was
almost exhausted. Some care must be excercised to
be sure that air was not aspirated into the tube, as
this produces voids in the metal column. Such
voids result in separation of the metal column
upon subsequent heating of the pipette to form the
electrode tip.
The microelectrode tip was formed in two steps.
The Sb-filled part of the tube was first pulled by
hand in a small flame, such as the pilot of a
bunsen burner, to a diameter of about 50-100/~m.
It may be necessary to pull more than one time in
the flame to obtain a sufficiently fine drawn segment. The Sb now forms a fine filament and must
be continuous in all parts of the segment. The fine
segment was then broken at 1-1.5 cm from the
point at which the tip of the electrode was to be
formed, and the end reintroduced in the flame at
an angle until a small hook is formed. The tubing
can now be mounted in a microforge, a weight

added to the hook and the tip formed in the usual

way. Microelectrodes which have been filled in the
way described above would not pull properly on
an automatic puller as the Sb tends to retract in
the tip. When formed on a microforge, the Sb was
usually flush with the tip and the glass cleanly
broken. On occasion, however, electrodes were
obtained with the Sb slightly (a few ~m) retracted
or protruding beyond the glass. In these cases, the
glass can be seen to be erratically fractured, but if
the tip is fine enough, they may be used reliably.
In general, however, a high yield of 1-2 /~m tip
diameter microelectrodes was obtained. The microelectrode was completed by melting the shaft
end of the Sb column in a small flame and introducing a clean bare copper wire of appropriate
dimensions for connection to the measuring
amplifier.

Electrode calibration
The electrodes must be calibrated before use as
they are not absolutely uniform in responsiveness
and, as pointed out by Fujimoto et al. [4], should
be rejected if they do not show at least - 4 5
m V / p H unit sensitivity or if they show any drift
during the calibration. The test solutions used here
were 50 m M Tris-maleate (Sigma), p H adjusted
with HC1 using a Radiometer model 28 p H meter
and a combination electrode. As pointed out by
several Sb electrode users [4,8,9] the relation between p H of the tested solutions and the potential
of the electrode is almost linear for p H from 3 to
10 with a slight change in slope at p H 7 (see
Fig. 1). Moreover, Hepes (Sigma) appears to be
equivalent to Tris as a buffering system for
calibrations while phosphate introduced a significant change in H+-senstivity of the antimony microelectrode [10].
The tracing shown in Fig. 1A, B and C are
recordings obtained by changing the bathing
medium with a peristaltic pump. It might be expected that the response time should be the same
for each change in pH, but in fact it can be seen in
these tracings that the time to reach steady state
becomes longer as the p H is enhanced. This slowing of the response is interpreted to be due to an
increased time necessary to achieve proton dilution as the p H is increased. For two reasons, first,
in the decreasing direction, steady state is more

339
my.

-5001

,,

0j -~

100G G

87! C .my

8.1 '

G-~en'q~/~

BB3528CM

1Glen

E sb

-- L_I--250

2rnin

pH

ef
Fig. 2. Experimental set-up for testing properties of antimony

7.

microelectrodes. The potential of the Sb electrode (Esb) was

recorded with a low leakage current operational amplifier. For
resistance determination, current was supplied bY a stimulus
generator (Gen.) through a 100 G~ resistor while current (I)
was measured using a second amplifier as an I~ V convertor
with a 1 Gf~ resistance connected to the chamber by an
Ag-AgC1 wire electrode. In order to reduce capacitance, the
voltage amplifier was mounted very near the Sb electrode. The
100 Gf~ resistor produced greatly slowed current pulses (see
Fig. 5).

/o

j.

6.

QJQ

-360

-3g0

-460

-~0

-s60mv

Fig. 1. Antimony microelectrode calibration Upper trace: Example of voltage recording obtained at different test solutions
from pH 3.8 to 8.7. Parts A and C correspond to the scale on
the left and B to that on the right. Solutions were changed
using a perfusion pump until a plateau was reached Lower

diagram: Calibration curve of the antimony electrode from the

recording above, The points may be jointed by two straight
lines with a small difference in slope occurring at pH 70.

For resistance measurement, and to study the effects of leakage currents in the electrode, a voltage
source was added between the input of the amplifier and ground, as shown in Fig. 2.
All recordings were made with a Brush 280 pen
recorder which has a full scale response time of 10
ms to a square wave input.

Results
rapidly obtained with the same flow conditions,
and secondly, if a large volume is used with the
perfusion system, oscillatory changes are seen due
to the movements of imperfectly mixed media in
the measurement chamber. The slowing of the
response with increased p H is therefore not considered to be due to a p H - d e p e n d e n t response time
of the electrode itself.
Measurement of the Sb electrode potential must
be done with an electrometer type of amplifier as
the resistance of these electrodes tends to be greater
t h a n 10 9 ~. We have found that a good quality
F E T * input o p - a m p having input-bias currents
less than one pA, such as the Analog Devices A D
52J or the Burr Brown 3528CM are quite adequate
even for very long term recordings of up to 5 h.

* FET, Field-effect transistor.

Electrode response time

For testing the response time, the electrode was

m o u n t e d in a micromanipulator which could be
made to drop by gravity through a height of about
3 cm. The drop carried the Sb electrode through a
p H discontinuity, created by floating a low density
(without sucrose) solution at one p H on top of a
higher density (with sucrose) solution of a second
pH. The b o u n d a r y between the two solutions can
be made quite sharp by drawing off interface
solution with a syringe and long needle until it can
be visualized by the differences in indexes of refraction. If the p H discontinuity is created in a
sufficiently long container centered on the axis of
the Sb electrode, the latter can be made to drop
very rapidly from one p H to another. In the set-up
used the amplifier was made to drop with the
electrode, in order to avoid flexing of the lead

340

wire, as this would introduce large capacitance

change voltage artifacts. In spite of this precaution, some artifacts are visible at the outset of the
drop and at the end when the micromanipulator is
abruptly arrested by a rubber pad (Fig. 3). The
inset in this figure shows that the interface between the two solutions is somewhat diffuse, being
about 8 m m for 100% change and about 4 m m
between the 10 and 90% limits. In this particular
case the microelectrode was dropped through 3 cm
in about 54 ms. The transit time was determined
by measuring the time between the release and
arrest artifact and knowing the distance of the
drop. The average velocity is then 55 c m / s , at
which the p H interface of 8 m m should have been
traversed in 15 ms, or if only the 10-90% limits
are taken, it is traversed in about 8 ms. If the time
course of only the most rapid change in potential
is measured, it is also about 8 ms and extends to
15 ms for 90% of full response. The same speed of
response was obtained when the change of p H was
from a higher to a lower value. When tested with
the same p H on both sides, no change in the
potential was observed on traversing the interface.
It can be seen that a slow change in Esb continues for many seconds after the electrode is
arrested (Fig. 3). This creep is thought to be due to
the time needed to reach an electrochemical equilibrium. In the worst case, the time constant introduced by the electrode resistance and input capacitance of the amplifier, input lead and electrode

should not exceed 100-200 ms ( R . C). For example, a 2 G O electrode resistance with a 10 pF input
capacitance gives a time constant of 20 ms which,
in turn, should give a response to better than 99%
in 120 ms if Esb were perfectly square.

Effects of currents in the electrode

Currents in the pA range in these electrodes
produced severe changes in the measured potentials owing to their resistances in the GfL The
resistances were found to depend on the p H in
such a way that negative currents (positive charges
entering the electrode tip) tended to decrease the
p H sensitivity while positive currents tended to
increase the sensitivity (Fig. 4). An Sb electrode
carrying a positive current can show a p H sensitivity greater than the theoretical value while those
with negative currents showed a smaller sensitivity. This effect is diminished as the resistance of

-50
I

-100

50
I

pA

100
I

150
; ~

. ~ p H 6
-200.

-300"

cr
200msec

pH 7

2//sec

mY
t

-450

o..*

-600

-400

pkl 6

1
5

J
10

I111"11

Fig. 3. Electrode response time to rapid p H changes. Ink writer

(Brush model 280) recording of Esb as the electrode is dropped
through a pH gradient. The inset shows the steady state Esb
obtained for stepwise movement of the same electrode through
the same gradient.

mV
Fig. 4. Effects of currents on Esb. Current voltage relation for
one electrode at three different pH values (e) and another
electrode at pH 6 (). The inset shows a typical recording
from which the I - V curves were obtained. For large negative
currents, the relation becomes markedly non-linear (see also
Fig. 5B).

341

the electrode decreased with increased size. However, for electrodes in the 1-2/~m diameter range,
the effect of current in the electrode was very
severe (see Fig. 5).
These measurements also show that the resistance of the electrode remains practically linear
with the current at a given pH but became more
non-linear at pH 6 than at pH 7 or 8. At zero
current, however, the potential obtained at a given
pH is very reproducible across electrodes. One
such example is shown in Fig. 4, where two electrodes of quite different resistances (0.9 and 1.8
G~2) give very nearly the same potential at pH 6
and zero current. This was a consistent finding for
all of the electrodes fabricated.
The resistance of newly fabricated electrodes

A
V"'

"1

r
r

:t-

.r
r

500 msec

Fig. 5. Dependence of the electrode resistance with time and

current. Oscilloscope traces of potential (V) and applied currents ( I ) for an antimony microelectrode just after immersion
(A) and after 80 min in the same test solution, p H 7.0, (B). The
resistance increase to about 3-times the initial value while the
baseline potential ( - 4 2 7 mV) did not change significantly
during the same period. Upward indicates positive potentials.
Calibrations as marked.

tended to increase with time in the calibration

solutions up to about 90 mn. The resistance increase with time was more pronounced in electrodes which had initial resistances in the hundreds
of Mf~ (see electrodes 8 and 10 in Table I). Electrodes with low initial resistance, less than 100
M~2, did not markedly enhance their resistance
(see electrode 3 in Table I). In spite of the large
range of resistance values, Esb was remarkably
constant with time, but the continued application
of current produced decreased potentials in some
electrodes. Electrode 7 in Table I showed a very
low potential ( - 3 2 0 mV) and a high initial resistance (1.3 GO) which changed very little with
time.
Polarization effects of currents in the electrode
are greatly increased after the resistance had increased. Initially, large currents in the electrode
produced only constant shifts in potential away
from the baseline value determined by the pH.
With the passage of time and the increase in
electrode resistance, smaller currents produced
time-dependent shifts in potential resembling a
polarization effect. These effects of current in the
electrode are illustrated by the superimposed oscilloscope tracings for the potential and the current
in Fig. 5. In the newly made electrode, the resistance is low and the potential change produced by
the pulses of current does not change during the
pulse (Fig. 5A). After 80 min, the resistance has
increased about 3-times and the potential continues to drift during the current application (Fig.
5B). This latter result indicates that the current
enhances the resistance of the electrode. It can also
change suddenly, as seen in the last negative going
trace in Fig. 5B. Such an effect of currents in the
electrode will be interpreted as shifts and jumps in
the indicated pH.

Intracellular recordings
Intracellular proton activity has been monitored
in oocytes from Xenopus laevis. For this purpose,
the cell was penetrated with an antimony microelectrode and a second one filled with 3 M KC1
for reference and voltage recording. In the present
experiments, internal pH was determined by subtracting the membrane potential (typically - 5 0
mV) from the Sb electrode potential (Fig. 6A and
B). Resting cytoplasmic pH was about 7.45 (Fig.

342
TABLE I
Sb ELECTRODE RESISTANCE CHANGES WITH TIME
Resistance of five antimony microelectrodes measured at 0, 30, 60, 90 and 150 min (one case) in a test solution at pH 7. The measured
resistances were normalized to the maximum value for each electrode and the means determined at the indicated times. The electrode
potentials, Esb, did not show any significant modification, except for number 7, which was initially abnormally low and began with a
high resistance.
Electrode resistance (GO)
Time (min):

Esb
(mY)

30

60

90

No
3
5
7
8
10

0.056
0.30
1.25
0.30
0.21

0.081
0.39
1.30
0.51
0.89

0.081
0.47
1.30
0.64
1.06

0.081
0.51
1.20
0.81
1.13

Mean normalization ratios

0.56

0.86

0.95

1.00

C02

100"/.

my

1-,-0
-20
0

mY

~t

~-'-~

-z,8o

-/-,60
-,'-40
-420

pHi
7.5

150

- -

0.65
-- -

413
- 422
- 320
-434
- 432
-

6C) which is in agreement with results obtained on

the same preparation using pH selective resin [11]
as well as in the egg stage using glass pH-sensitive
microelectrodes [ 12].
As illustrated here, external perfusion with a
solution saturated with 100% CO 2 induced rapid
and reversible cytoplasmic acidification. In Fig. 6
pH was shifted by about one unit which was in the
range of results obtained in other preparations
[ 11,13,14]. Moreover, delayed overshoots of 0.1-0.3
pH units were also seen after washing (data not
shown). In view of the rapid response of the
electrode, the rise time shown in Fig. 6C is probably due more to the time to change the solution in
the recording chamber than to the CO 2 diffusion
across the oocyte membrane. This ambiguity may
be resolved by having a second antimony electrode
in the bath to monitor the rate of pH change
outside the cell.

Conclusions
60sec

6.5

P'ig. 6. Intracellular pH monitored with an antimony microelectrode. Potentials recordings from an electrode filled with KCI
(A) and an Sb electrode (B). C: point by point subtraction of A
from B plotted in units of pH, which at rest was 7.45. Superfusion for 90 s with a solution saturated with 100% CO 2 (bar)
induced a large cytoplasmic acidification (0.8 pH unit) which
was reversed with washing.

There are two main results of this study of the

antimony pH-sensitive microelectrode properties.
First, the speed of response of this electrode may
have been predictable by the fact that Esb is an
electrochemical potential which should be determined directly by the hydrogen ion activity at
the electrode tip. The 100% response time of a few

343

seconds was also pointed out by Fujimoto et al. [4]

but they did not show any direct recordings. The
recordings presented here show that for large shifts
in pH, time courses in ms may be expected using
this type of electrode if care is taken to keep the
input capacitance in the low pF.
Secondly, currents in the pA range are sufficient to significantly modify the pH sensitivity.
This point may explain part of the poor reputation
of the Sb electrode [1]. The same electrode will
give different calibrations with different electrometers depending on the magnitude and direction of
the leakage current. The same effects will be obtained if resistive paths to ground, in the order of
GfL exist in the input circuit because of the large
potentials produced by the electrode. At pH 7, Esb
is about -420 mV so that a current of 10 pA is
obtained with a shunt path of 42 G~2 as may be
obtained with poor insulating materials.
These electrodes have been used for intracellular pH determinations in crayfish giant axons [15]
and in Xenopus laevis oocytes. The agreement of
measurements with those reported using other
methods [3] indicates that the antimony microelectrode can be used reliably for intracellular pH
monitoring. Finally, its rapid response time may
make it useful in studies of the dynamics of cytoplasmic pH change.

Acknowledgements
We wish to thank Mrs. J. Nicolet for her helpful
assistance throughout this work. This work has
been partially supported by DGRST grant No.
81-E-0576.

References
1 Thomas, R.C. (1978) in Ion Sensitive Intracellular Microelectrodes: How to Make and Use Them (Thomas, R.C.,
ed.), pp. 1-110, Academic Press, London, New York, San
Francisco
2 Ives, D.J.G. and Janz, G.J. (1961) in Reference Electrodes:
Theory and Practice (Ives, D.J.G. and Janz, G.J., eds.), pp.
1-651, Academic Press, London, New York
3 Roos, A. and Boron, W.F. (1981) Physiol. Rev. 61, (2),
296-433
4 Fujimoto, M., Matsumura, Y. and Satake, N. (1980) Jap. J.
Physiol., 30, 491-508
5 Matsumura, Y., Satake, N. and Fujimoto, M. (1980) Jap. J.
Physiol., 30, 509-528
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Physiol., 30, 671-687
7 Satake, N., Matsumura, Y. and Fujimoto, M. (1980) Jap. J.
Physiol., 30, 689-700
8 Bicher, H,I. and Ohki, S. (1972) Biochim. Biophys. Acta
255, 900-904
9 Vieira, F.L. and Malnic, G. (1968) Am. J. Physiol. 214,
710-718
10 Green, R. and Giebisch, G. (1974) in Ion Sensitive Microelectrodes (Berman, H.J. and Hebert, N.C., eds.), pp. 43-52,
Plenum Press, New York
11 Ammann, D., Lanter, F., Steiner, R.A., Schulthess, P.,
Shijo, Y. and Simon, W. (1981) Anal. Chem. 53 (14),
2267-2270
12 Webb, D,J. and Nuccitelli, R. (1981) J. Cell Biol., 91,
562-567
13 Thomas, R.C. (1974) J. Physiol. 238, 159-180
14 Boron, W.F. and Deweer, P. (1976) J. Gen. Physiol. 67,
91-112
15 Giaume, C. and Korn, H. (1982) Neuroscience, 7 (7),
1723-1730