Abstract: Carbon-fiber microelectrodes were used to electrochemically
measure concentrations of oxidizable neurotransmitters, serotonin,
epinephrine, and norepinephrine. The electrodes consisted of a carbon
filament surrounded by a glass capillary tube. The carbon filament was
connected to a patch clamp amplifier to set the filament at a certain voltage
and to measure current flow, which increased during oxidation. This was
done both with a triangle wave, cyclic voltammetry, and a set potential of +5
Calibration curves were done with known
and .6V, amperometry.
concentrations of both epinephrine and serotonin in vitro. In vivo recording
were performed in Pleurobranchea pedal ganglion and NIE-115
Neuroblastoma cells. Epinephrine behavior was close to theory with a one to
one relation between increase in I and [Epil, but the response to 5-HT was
not as strong. The electrodes measured 90 uM (400 pA by amperometry
release of serotonin from the Pleurobranchea pedal ganglion. Readings were
also taken from NIE-115 cells which showed 1 to 100 uM (1.6nA)
norepinephrine release after stimulation with carbachol, a non-oxidizable
analog of acetylcholine.
Introduction: Carbon-fiber microelectrodes are, and have been for years,
used in electrochemistry. The method has great potential for looking at
biological problems because it has fantastic speed, within milliseconds, and
sensitivity, detection down to 100 nM (Wightman, 1981). It has even be used
to measure single vesicle release (Chow et al., 1992 and Wightman, 1991).
The technique takes advantage of the fact that amine-comtaining
neurotransmitters are easily oxidizable. All catecholamines for instance,
oxidize in a two-electron process, shown for epinephrine in Figure 1.
Because the two released electrons are constant, they are, theoretically, a
measure of the amount of oxidation that has occured. The carbon-fiber
electrode takes advantage of this on a small scale and uses a 8 micron
diameter carbon-fiber. As shown in Figure 2, when the voltage potential of
the electrode, as locked in by a patch clamp amplifier, is above the oxidation
potential of the neurotransmitters, which is different for each substance, the
carbon filament oxidizes the neurotransmitter. The higher above this the
oxidation point the potential is set, the greater the rate of oxidation at the
electrode surface.
When a range of set voltages for the carbon-fiber is scanned, a leak and
capacitance base current causes a fluctuation of current, higher when it
increases and lower when it decreases. Above +1 V water is oxidized causing
a large peak. To determine the exact current for a disk shaped electrode
which is totally irreversible (assuming low concentration so that the oxidized
product diffuses away before being reduced) the equation for the current can
be solved to:
i= Covl2k
v1/2 is the scan rate and because i = C dv/dt for any capacitor, like this
electrode, the higher the scan rate the higher the background current from
capacitance. Co is the concentration of oxidized substances. K is a factor
that takes into account a number of different factors, such as the geometry of
the carbon-fiber surface and the nature of the oxidation reaction occurring.
The relation to current and concentration in this equation is direct for any
given voltage (Bard and Faulkner, 1980, 222). When the carbon-fiber is set
at a certain potential, the situation is similar to when one particular voltage of
the whole range scanned in cyclic voltammetry is examined. Concentration is
important because the only way new oxidizable molecules get to the carbon-
fiber surface is by diffusion which is driven by the concentration.
Not all catecholamines are the same, and serotonin presents a special
difficulty because its oxidized form creates a film over the electrode which
reduces the electrodes sensitivity (Wightman, 1988). It can be reduced by
scanning at very high scan rates (200V/s) which unfortunately also involves a
lot of background current, making it difficult not to saturate the patch clamp
amplifier.
The next part of this study was to use the microelectrodes to measure
neurotransmitter release in biological systems. Two systems were chosen.
First, The pedal ganglion of the Pleurobranchea is believed to secrete
serotonin (Rhanor Gillette, personal communication) which is supported by
the production of serotonin by the pedal and abdominal ganglia (Wilbur,
1985), and the NIE-115 mouse neuroblastoma cells, which contain
norepinephrine (Herken, 1982).
Materials and Methods:
Electrode Fabrication:
The carbon-fiber electrodes were made by a standard method.
(Fishman, 1992). The carbon-fibers were spread onto a clean piece of paper.
Figure 3A is a picture of carbon-fibers. Smoother paper worked better. A
single fiber was separated with the finger tips. Multiple fibers were be fairly
easily visually distinguished from single ones. Two fibers together can be
separated by rolling at the tip or the middle. Fibers were selected that were
as long as or longer than the capillary tubes. A vacuum pump (Air Cadet,
Model No. 7530-40) was attached to a glass capillary tube (Work Precision
Instruments, Cat No TWISOF) and used to aspirate a single carbon filament
whose tip was overhanging the paper 2mm. The far end of the fiber was held
to prevent the whole fiber from being sucked in. Using the vacuum pressure,
the fiber was brought all the way through the capillary tube.
The capillary tube was then pulled in an electrode puller (David Kopf
Instrument Vertical Pipette Puller, Model 700C, modified by Professor
William Gilly) with heat set at 47. The carbon-fiber that separated the two
electrodes was cut and the electrodes were removed. The next step was to
dip the tips into epoxy (Epo-tek 301) for ten minutes while it filled the tip.
Four hours in the oven (NAPCO, Model 5830) at 65°C was adequate to dry
the epoxy, making a working electrode, Figure 3B. There was a differences
between different electrode pullers. The Narishiga PE-2 would not pull
electrodes that would fill with epoxy after pulling.
Electrodes were painted with silgard and then heated until dry. The
electrodes made this way worked normally and were made much more
quickly.
The last step was to cut the carbon-filament very close to the end of
the glass of the capillary tube. A razor blade was used to cut the carbon
fiber. This was done immediately before experimental use.
Testing the microelectrodes:
The electrodes were tested in vitro. Baselines were tested in distilled
water with 150 mM NaCl. A ground, a bare silver/silver chloride wire, was
put into the solution. The electrode was connected to a headstage (Warner
Instrument Corporation, 5C-501) of patch clamp amplifier. The output
current was filtered (Frequency Device, 902) at 20 hz. The command voltage
was provided by Exact, model 119 Function Generator. Constant potentials
for amperometry were provided by a de current source. The current and
voltage were viewed on a Nicolet 3091 digital oscilloscope. Electrodes were
simply put into the pools with varying known concentrations of
neurotransmitter for testing. To prevent premature oxidation, the solid
compounds were stored in a freezer in a light opaque container. When the
epinephrine and serotonin solutions were measured out, they were added to
150 mM NaCl solution that had been bubbled for 1 minute with 99.999% N.
(Liquid Carbonic) and sealed with Parafilm. As soon as the compound was
added it was again bubbled with N2 and sealed. For a portable N2 source,
balloons were filled with N2 and fitting with a rubber tubing adaptor and a
needle.
Epinephrine dose response calibration:
The electrodes was positioned in the bath and solutions were changed
by using the profusion device. The carbon filament was kept at +.5V and
tested at a range of concentrations. The order was: baseline with O mM
epinephrine, then stepping up concentrations, and then ending with another
wash at 0 mM epinephrine. The current and the command voltage were
measure on tape recorder (Tanberg Instrumentation Recorder, Series 115).
The information was then digitized.
Measurements in the Pleurobranchea pedal ganglion:
Before the experiment, a dose response calibration was done with
creatine sulfate serotonin. Concentrations of 10-8 to 10-3 serotonin were
tested. Between each serotonin concentration, the baseline was again
established by returning the electrode to a pool of without serotonin. The
difference was measured directly off of the oscilloscope screen. A suction
electrode was used to attach to a nerve bundle so that Grass SD9 Stimulator
could stimulate the whole cluster. A microelectrode was placed in one of the
ganglion's nerves to measure activity. The carbon-fiber microelectrode was
placed under a group of cells. The four track tape recorder was used to
record the responses of the current and command voltage for the carbon
fiber microelectrode and the membrane potential which were transferred to a
chart recorder for analysis (Gould Recorder 220). During the experiment the
table was tapped.
Testing NIE-115 neuroblastoma cells:
This experiment was done on a patch clamp set-up (List-Medical Patch
Clamp LM-EPC 7 amplifier and LIM-EpC 7 5/n 583644 headstage) and was
filtered at 3khz (Frequency Device, 902 LPF). A DAT tape recorder (Sony
High Density Linear A/D D/A tape recorder) was used to record the
amperometry data at +.6V. The same type of oscilloscope was used. A
gravity perfusion system was used to change concentrations. A calibration
was done for epinephrine both at the beginning and the end of the
experiment. The effects of 1 mM carbachol alone were also tested. The
electrode was positioned with a very long carbon filament so the carbon
filament was resting on a group of NIE-115 mouse neuroblastoma cells
which had been raised in culture (Mathes, 1992). The cells were kept normal
external saline (Mathes, 1992). Carbachol was then added.
Results:
Testing electrodes:
The behavior of typical carbon-filament microelectrode under cyclic
voltammetry is shown in Figure 4 if it is viewed with both current and
voltage on the Y axis and time on the Xaxis. The voltage trace is the triangle
wave. The current is the trace showing the curve. Each microelectrode will
show a different current trace, but the basic shape should be the same. It
should show extremes at both ends of the current trace. It should also show
another peak as the current increases past zero in the presence of an
oxidizable substance, as it does in the lower trace of Figure 4.
A working electrode in cyclic voltammetry should look like Figure 5A
if it is graphed xy with current the y axis and voltage the x axis, a
voltammogram. Each electrode again has a different basic shape. The bettei
the electrode and the slower the scan rate the thinner the leak and capacitance
is, which causes open middle to be reduced. A functional electrode also has
to show another peak when an oxidizable material is added that should be at
the positive side of the increasing voltage, the upper right of the curve. The
peak increases and moves to the right as the concentration increases. At high
concentrations, as the 25mM in Figure 5A, other differences can be seen.
There are two troughs, the first smaller than the other. Figure 5B is graph of
computer subtraction the baseline from the after oxidation trace of three
different epinephrine concentration's voltammogram and shows these other
peaks very well.
To eliminate leak and capacitance currents a baseline was subtracted
from the voltammetric traces. Voltage and current were digitized (the
computer programs used were Clampex version 4.10 with Amp and Clampfit
Version 5.5 from Axon Instruments on an ZTS 386 computer with the A/D
input designed by Stuart Thompson) and the base OM epinephrine was
subtracted from the curve with the various epinephrine concentrations. This
was done by starting with the minimum point taking one whole cycle of the
curve to get the two traces in the same phase.
The level of the current corresponds almost directly to the
concentration. Figure 6 shows this with the current trace both before and
after a SmM increase in concentration. The epinephrine calibration was done
this way. The plot of concentration and current is Figure 7A. The plot of the
log of concentration and log of current is Figure 7B. The best fit linear fit for
this line (Cricket Graph 1.3) was y-5.43 + 0.86x which makes the power law
for this relationship y- 269153.47x0.86 (x-M, y-PA). Figure 7C is a plot of
log of concentration and log of current for serotonin. This line has a slope of
y=3.17 +0.14x (Cricket Graph 1.3). This was the best serotonin calibration
that was obtained. It was preformed before the Pleurobranchea experiment.
Figure 9A shows the cyclic voltammetry during the Pleurobranchea
experiment. This change occurred without stimulation. Figure 9B shows a
trace from the same experiment. The jump in amperometry corresponds to a
physical shock. The jump was 400 pA and decayed back down to the
baseline.
Figure 9A does not give quantitative data, because a voltammetry
calibration was not done before the experiment. However it does raise and
answer some interesting questions. First it does not correspond to any of the
nerve stimulation that were given to the ganglion. It was a completely
spontaneous event, independent of activity in the one cell of the cluster whose
membrane potential is measured on the lower trace. However the current
trace is not ambiguous. It shows a dramatic increase in current and a very
definite peak at the raising positive voltage. No apparent harm was done to
the cells when shaken for the cell with the current clamped microelectrode
continued to spike normally. Figure 9B gives more quantitative data but has
more questions about it's validity. The signal was triggered by shaking the
table, but the signal is what was expected for release during amperometry.
Based on the calibration in Figure 7C 90 uM serotonin would give this
response.
The mouse neuroblastoma showed this same pattern, but in a more
controlled fashion. After the carbachol, which is not oxidizable, was added to
the NIE-115 cells the current rose dramatically from baseline by 1.6nA and
then slowly decayed as seen in Figure 10. When carbachol was added
without cells there was no change in the baseline. What is remarkable is the
slow time of the rise if what is measured is vesicle release. However, The
carbon-filament used was long and lay over a cluster of the cells. Therefore
the data represents the sum of the release from a number of cells which
would account the slow overall release. The calibrations were off and
epinephrine, not norepinephrine, was used, but the concentrations recorded
should correspond to norepinephrine levels between 1 and 100 UM.
Discussion:
The major finding of this study is that electrochemical detection of the
catecholamine neurotransmitters serotonin, in Pleurobranchea pedal ganglion,
and norepinephrine, in NIE-115 neuroblastoma cells, is possible.
The first lessons is that there are four basic types of electrodes.
Diagrams of the first two types are in Figure 8. Electrodes of the first type
were broken. The current saturated in the direction of the voltage, positive
for positive voltages and negative for negative voltages, except around zero
where they changed. The second group (8B) were electrodes that had no
carbon-filament exposed. They showed a flat trace until the oscilloscope gain
was increased to the maximum and then showed a parallelogram trace with a
negative current. The third group showed normal voltammograms, but
showed no response to epinephrine. The fourth group were functional
electrodes which showed normal voltammograms and showed an increase of
current with epinephrine. Occasionally the functional electrodes would show
a trace like the third trace on Figure 8. This was simply a problem of the
gain being too high (.5pA/mV instead of .IpAlmV) for that particulai
electrode. Higher gains often gave even more problems because of
mismatched impedance between the electrode and the headstage.
The calibration for epinephrine is very promising. Figure 7B has a
slope near 1,.86. This is close to theory. Even so I would advocate a dose
response calibration before and after each experiment to see any deviations
from normal because each electrode is different and use changes its response.
As the electrodes are used they get dirty (Fishman, 1992) which reduces their
sensitivity. The response of a dirty electrode would be less current change for
a given concentration change which is what was observed.
10
Figure 7C is more troubling. The response to serotonin is much worse
than expected. The slope is a seventh of the theoretical value. It has been
stated that after being oxidized serotonin forms a film over the carbon-fiber
(Wightman, 1988). Again this dirtying of the carbon-filament surface results
in the slope of the log graph less than one. The value, .14, indicates a very
dirty electrode. This loss of sensitivity with corresponds with experience
with serotonin during other attempted calibrations. For example, an electrode
started with strong response of 900 pA from 10-3M, and yet when the
calibration was finished, the electrode showed no change at 10-2M.
It is difficult to know exactly what compound's oxidation is being
recorded using this technique. But I feel secure in the correct identification of
the compounds in these experiments. In the Pleurobranchea experiment, the
first trace is serotonin. It is released without any outside disturbance of the
cell, and it is a single clean peak in the oxidation which suggest that only one
oxidizable species is involved. The amperometric measurement is more
difficult to identify as secreted serotonin. Although is seems very unlikely, it
could be simply the result of cells being lysed and spilling their oxidizable
contents onto the electrode. However, the cyclic voltammetric measurements
clearly show that the cells are able to release serotonin. If the electrode really
had broken into a cell, the oxidation would occur more rapidly, and the decay
should have been slower if it was trapped in the cell's contents.
Norepinephrine occurs in NIE-115 cells ten times as much as the
other catecholamines found, dopamine and serotonin. Therefore it seems
likely that it is the major component of the release.
The release that is linked to carbachol is without action potentials, so
this release occured without spikes. Carbachol is detected by the muscarinic
acetylcholine receptors which in this preparations does not cause action
11
potentials. The conventional pathway-action potential, calcium influx, and
then secretion—is bypassed. This shows that the calcium influx may be
sufficient to get norepinephrine release.
The concentration of transmitters released in these systems has not
been measured before. Both concentrations are in uM range and localized in
a very small area. This is a very powerful signal even taking into account the
artificial nature of the stimulus. Wightman reported total concentration of
catecholamine release from bovine chromaffin cells of 10 uM (1991).
Serotonin receptors have very high affinities, upper nM concentrations, for
serotonin (Wang and Peroutka, 1988). This means that there would be a huge
detection of these signals. Again I must state that these concentrations are
high, but the exact level I am unsure of, because the calibration for the two
experiments were poor.
I would like to stress that these results are largely suggestive. They
raise some interesting questions about the biological systems which were
observed. However, they were done in this study to examine the behavior of
the carbon-fiber microelectrodes in biological systems. In this respect, they
were complete successes. The phenomena exhibited, whatever their cause,
were very much what was expected for both voltammetry and more
important amperometry in two different systems.
There several areas yet to be explored. Reliable dose-response
calibrations is needed before anything else. The technique of cutting the
carbon-fiber very close to the glass or polishing the surface also needs to be
improved to improve detection. Now that this procedure is working, the
options are almost endless.
12
Acknowledgements: I am going to make this section all together too long
because I will attempt the impossible and thank all those who deserve it. I
will undoubtedly forget some of those who need it (like Mom who is always
forgotten at times like this) I apologize profusely, the mistake was not because
I have forgotten your contribution but is entirely the fault of my sleep debt.
First for the scientific thanks, both Rhanor Gillette, Rob Swezey, and
Harvey Fishman gave me scientific aid when I requested it. In fact, Harvey
Fishman saved my life and gave me the key that allowed me to get it
working. I would like to thank them all for helping a lil' undergrad as he
floundered his way through his project. I would also like to thank Gilly for
the stream of equipment he lent me (even after I started breaking his stuff)
The first person who needs to be thank is Stuart, who if he had any
sense, would have kick me out of his lab the first time I walked in the door or
at least when I screwed up with the electrode puller after he spent 15 minutes
telling me not to screw up. Thanks for giving me a change to screw up again
(and again, and again....). Stuart was also "totally key" in getting both the
technical and theoretical aspects of this project working. Sorry I didn’t listen
and follow more than I did.
Sam Wang needs to be thanked not only for scientific help, but more
importantly for trying to give me an education in bad music and helping me
keep my sanity. I also would like to thank you for not just doing this project
yourself (although you would certainly have done a better job). I pray that
you'Il find your blonde (my addition) tortured artist very soon.
Iwould also like to thank Chris for getting me to experience first hand
how gross coffee really is and showing me the terrors of married life and
those totally killer NIE-115 cells.
13
Now for the random thanks. My housemates and lab partners need to
be thanked to putting up with my insanity-"overwork leads to all forms of
5
s. 1 d like to thank Coke giving me the calories and caffeine to
metal disorder:
survive this quarter. I'd like to thank my body for putting up with all the
terrible things l've been feeding it. Most important l’d like to thank 107.1
rocks and 94.5 who I listed to almost continually (except 5 hours for sleeping
some Garth Brooks, Right Said Fred, and a little Debbie Gibson). To them I
owe for holding what little sanity I have left together. I shall miss 107.1 more
than anything else about Monterey.
My final thanks is to my father. All those nights of reading me to sleep
from Crucible: The Story of Chemistry had some effect after all. Thanks for
making me what little of a scientist I am.
Literature Cited:
Bard, Allen and Larry Faulkner. Electrochemical Methods:
Eundamentals and Applications. John Wiley & Sons, Inc.:New York, 1980.
John E. Baur, Eric W. Kristensen, Leslie J. may, Donna J. Wiedemann,
and R. Mark Wightman. 1988. "Fast-Scan Voltammetry of Biogenic
Amines." Analytiçal Chemistry. Vol 60, 1268-1272.
Chow, Robert H., Ludolf von Ruden, & Erwin Neher. 1992. "Delay
in vesicle fusion revealed by electrochemical monitoring of single secretory
events in adrenal chromaffin cells." Nature. Vol 356, 60-63.
Fishman, Harvey. Personal Communication. 1992. Stanford University
Chemistry Department.
Herken, Hans. et al. 1982. "Mouse Neuroblastoma clone NIE-115: A
suitable model for studying the action of dopamine agonist on tryosine
hydroxylase activity." Biological Pharmacology. Vol 31. No. 7, 1279-1282.
Mathes, Chris. M. 1992. "Intracellular calcium release in NIE-115
neuroblastoma cells is mediated by the MI muscarinic receptor subtype and is
antagonized by MCN-A-343." Unpublished. Accepted Brain Research.
Peroutka, Stephen and Samuel Wang. The Serotonin Receptors. The
Humana Press: Clifton, NJ. 1988.
Wightman, R. Mark. 1981. "Microvoltammetric Electodes.
Analytical Chemistry. Vol 53, 1125-1134.
Wightman, R., J. Jankowski, R. Kenedy, K. Kawagoe, T. Schroeder, D.
Leszczyszyn, J. Near, E. Diliberto, Jr., and O.Viverous. 1991. "Temporally
resolved catecholamine spikes correspond to single vesicle release from
individual chromaffin cells. Proc. Natl. Acad. Sci. U.S.A. Vol. 88, 10754¬
10758.
15
1985.
Wilbur, Karl. The Mollusca. Vol 8. Academic Press, Inc: Orlando Fl.
Figure Legend:
Figure 1-The oxidation of the catecholamines occurs by this general
two electron process which is shown here with epinephrine.
Figure 2-The microelectrode is given a potential by the amplifier. At
an adequate potential, the fiber oxidizes the epinephrine in contact
with it. This results in two electrons being transferred to the carbon
filament which is detected by the amplifier and recorded as higher
current.
Figure 3- A) A photograph of the carbon-fibers. B) A photograph of
a microelectrode.
Figure 4-A picture from a carbon-fiber microelectrode of a display of
the voltage and current on the y axis and time on the x axis. The
voltage is a triangle wave which correspond to the potential of the
carbon-fiber. The current is the current flowing through the carbon¬
fiber. The upper trace shows the background current. The lower
trace shows the current while oxidation is occurring.
Figure 5-A) An xy plot of voltage and current, voltammogram. The
inner trace is the background. The outer trace is in the presence of
25mM epinephrine. The additional current is from oxidation of
epinephrine. B) This is a voltamogram with the background
subtracted. There are three different concentrations here. The
electrode shows no change at O.0lmM epinephrine. O.ImM shows a
17
very distinct peak. ImM has a very large response. In fact, the
current has saturated the amplifier so the top of the peak is
flattened.
Figure 6-Amperometric response to a 5mM increase in epinephrine
concentration.
Figure 7-A) A graph of the amperometric current vs. concentration of
epinephrine. B) A graph of the log the concentration and log of the
current for epinephrine with the baseline subtracted. It has a slope
of .86. C) This is a graph of the concentration and log of the change
in current for serotonin. It has a slope of .14.
Figure 8—A) is the xy plot of voltage and current for a broken
electrode. The current saturates the amplifier for almost the entire
trace. B) An xy plot for an electrode with the carbon-fiber missing or
covered. The current is very small and negative, and the curve it
makes is a very angular shape. C) A normal electrode at too high
gain. When the gain is reduced the trace looks like Figure 6A.
Figure 9—Electrochemical measurement of serotonin in pedal
ganglion of Pleurobrachea A) The trace is the current with cyclic
voltammetry. The first half shows the baseline current flow. Then, a
change occurs and the current increases and changes its shape. Next,
the gain of the amplifier was turned down to prevent saturation.
After the change, an additional peak can be seen that corresponds to
oxidation at the carbon filament. B) The increase in amperometic
18
current corresponds to a mechanical stimulation. The voltage of the
electrode was +.6V.
Figure 10—Two digitized scans overlaid. The lower is the baseline
current done with the electrode in the bath resting on the NIE-115
neuroblastoma cells with a holding voltage of +.6V. The second,
higher, is current after carbachol was added.
CH5
HO-CCh
NH

OH
OH
Epinephrine

Figure
CH
— NH
HO-C-CH.

o-
Electron
Acceptor
0-

CH5
HO-C—CH-NH
Oxidized Epinephrine
1.
CI

Na
epi
Carbon
Filament
at +5V
epi
Na
Depletion Lager
Figure 2
To Amplifier
Ag/AgCl
Na'
CI
epi epi
Na
An epinephrine
molecule being
oxidized
Na
CI
Ground
Ag/AgCl
C1
B)
Figure 3
OM
25mM Epi
igure 2
5 sec
50 n4
B,
A)
18.0
16.0 -
14.0 -
12.0 -
10.0 -
8.0 -
6.0 -
4.0 -
2.0 -
O.0
-2.0 -
-4.0 -
-6.0
-8.0 -
-10.0 -
-12.0 -
-14.0 —
-16.0 —
-1.2
Figure 5
10nA
1V




Soførtst





—0.4
-0.8
0.4
O.8
Voltoge (V
1mM Epi
1mM
OimM
1.2
Figure 6
200 pA
sed
0
A


C
Figure 7
3+
2.8 —
2.6 —
2.4 -
2.2 -
1.8 -
1.6 -
1.4 -
1.2 -
0.8 -
0.6 -
0.4 -
0.2 -
o
Of+O0f-OBE-OTE-ORE-OSE-OSE-ORE-OSE-OSE-OOE-OGE-OAE-OREOSE-O3-OD-00
Concentration on Epl (M)
3.5 -
2.5 -
1.5 -
0.3-
o +
95 4
-6.7 -8.3 -8 -5.7 -5.3 -3 -4.7 -4.3 —4 -3.7 -3.3 -3 -2.7 -2.3 -2
Log of Concentration of Epl (M)
274 —
272 -
2.7 -
268 -
266 -
264
262
258 -
236 -
2.54
252 -
248 -
246
244 +
A
—
o B

Figure 8
—
Voltage
120
zero current
100my
below zero current
C
J


(
2
Current (pA)

S