Axon Instruments, Inc.
Marketing & Scientific Applications
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Phone (415) 571-9400 Fax (415) 571-9500
e-mail: tech@axonet.com
Configuring Axon Instruments Hardware and
Software to Collect Cyclic Voltammetric Data
Introduction
Cyclic voltammetry has been used extensively to study the neurochemical kinetics of dopamine release and
reuptake in a number of in vivo and in vitro preparations. The technique is quite simple. A controlled potential
waveform is applied to an electrochemical sensor and the current which is generated at the sensor is recorded.
Traditionally, a linear waveform (which is described in greater detail below) is applied to the sensor. For
biological applications, the sensor is typically a carbon-fiber microelectrode. The applied potential, which appears
at the tip of the electrode, results in the oxidation of an electro-active species (e.g. dopamine) in solution. The
current which is derived from the measurement is a complex combination of chemical kinetics and molecular
diffusion which is relatively specific for differ ent chemical classes of compounds. In short, the technique generates
a chemical fingerprint for each compound of interest. The technique becomes useful for neuroscientist because
repetitive measurements of these fingerprints can be made rapidly (as fast as 5-10 ms per measurement), and the
current which is derived from the oxidation (or reduction) of these compounds is directly proportional to
concentration.
Voltammetry is an example of an episodic voltage clamp experiment. The term episodic is used to describe any
technique in which the data is sampled at fixed time intervals (as opposed to continuous or gap-free sampling). In
the example which follows, real time (100 ms resolution) detection of dopamine is performed. The potential at
which dopamine (and other catechol-containing species such as epinephrine, norepinephrine) oxidizes is
approximately 0.15 V vs. the silver/silver chloride reference potential. In order to accurately measure the
voltammetric response of dopamine in solution, the sensor is poised at a reducing potential between measurements
and scanned to more oxidizing potentials to generate the electrochemical fingerprint. Sample introduction is
synchronized with data collection using the user list feature of Clampex to control a digital port output. This type
of control could be used to trigger a flow injection device or other external devices such as a picospritzer or
stimulator. Once the data is collected, analysis of the data is accomplished using Clampfit.
Instrument Configuration
Typical configuration:
Equipment: CyberAmp (320 or 380), GeneClamp 500, Digidata 1200.
Preamplifier: CV-5-100 M (Connected to Headstage #3 input).
Software: Clampex for data collection and Clampfit for data display.
Connections: The following connections should be made with the hardware listed above.
Potential:
DAC#0out = CA#1in
CA#1out = E#3in (200 mV/V)
E#3out = ADC0in
Current:
I3out (NOT Scaled) = CA#2in
CA#2out = ADC#1in
Where:
in and out subscripts indicate an input or output connection.
= - Indicates a connection.
DAC#n - Digidata 1200 digital-to-analog converter channel number n.
CA#n - CyberAmp channel number n.
E#3 - GeneClamp 500 electrode potential input or output.
ADC#n - Analog-to-digital converter channel number n.
Application Note #4
December 1994
Printed in U.S.A.
Page 1 of 4
Software Configuration
Clampex is used to define the potential waveform applied to the electrode and to collect the resulting data. In
addition, digital lines to sample injection devices, such as a picospritzer, can be configured and be synchronized
with the data collection process. The following sections describe the Clampex configuration information required
to run cyclic voltammetry. Bolded letters indicate user input. Figures in the text show the details of each screen
involved in configuring the software. Once you have configured your system properly, you should save your
settings in a parameter file.
Under Configure, Hardware:
Before data can be collected, the hardware must
be specified. The figure to the right is a typical
configuration for the specified Axon hardware.
Note the configuration on your system may be
different.
Under Parameters, Acquisition:
In this example, the potential applied to the
working electrode (sensor) is ramped in a linear
fashion with respect to time. This is analogous to
a voltage clamp experiment.
In this
configuration the Digidata 1200 samples data on
channels 0 and 1 at 0.1 second intervals and
repeats the measurement 100 times. With the
hardware configured as indicated above, the
applied potential will be returning on A/D
channel 0 and the current on A/D channel 1.
Each episode consists of 256 data points/channel.
Under Parameters, Waveform:
This section shows how to define a potential
ramp from -0.4 to 1.4 V and back to -0.4 V. The
voltage applied to the electrode is generated with
DAC 0 and applied to the E#3 (200 mV/V) input
of the GeneClamp 500.
To calculate the
potential scan rate, note that 124 points are used
to scan a 1.4 volt range. As defined in the
previous window, each point represents 24 µs
and thus the scan rate is 470 V/s (1.4 V /(124
points * 24 µs/point)).
Application Note #4
December 1994
Printed in U.S.A.
Page 2 of 4
Under Parameters, Scaling:
For the GeneClamp 500, the voltage applied to
the electrode is scaled by a factor of 200 mV/V.
Since the recorded voltage is a factor of five
times greater than the applied voltage, the
scaling of the signal is done by setting the
acquisition scaling factor on channel zero. The
GeneClamp BNC connection I3 out carries the
current from the electrode.
This current,
measured at DAC 0, is scaled by a factor
determined by the preamplifier used. For the
CV-5-100 M, the gain is 10 nA/V (or 0.1
V/Units). Note that the output channels have
been scaled for correct output.
Under Configure, CyberAmp, Settings:
In cyclic voltammetry, a true linear ramp
is employed.
Using the CyberAmp, the
programmed waveform can be low-pass filtered
to closely resemble a true linear waveform. This
can be done empirically by setting the waveform
filter (Channel #1) so that minimal distortion of
the recorded triangle wave is observed. Under
these conditions, a cutoff frequency of 6000 Hz
can safely be employed without distortion of the
signal.
Under Parameter, Userlist:
To precisely control the sample introduction, a
userlist is defined. The userlist is used to control
the digital output lines of the Digidata 1200. As
defined below, background scans are collected for
a 0.5 seconds and a signal is introduced for 3.4 s.
The final input of the userlist forces the output of
the digital line back to its initial value of zero.
Note that data is collected for a total of 10
seconds (100 episodes at 0.1 seconds) to monitor
the electrode response after the termination of the
event triggered by the user list.
Application Note #4
December 1994
Printed in U.S.A.
Page 3 of 4
Collecting Data
Under Configure, Fastloading:
In this example, the background current is first
allowed to stabilize for 100 cycles and then the
data, with sample injection, is performed. Two
parameters sets are used. The first, CV01.BPA, is
saved with the user list disabled. CV02.BPA is
saved with the user list enabled. Fastloading is
used to sequen tially run these files and collect the
data set of interest (under control of CV02.BPA).
To activate the conditioning and collection scans,
Ctrl-F1 is used. Note the link allows CV02 to be
loaded and the data collected.
With a 10 µm carbon-fiber disk electrode, you
should expect to see a stabilized current response
similar to that shown on the right. When the
program is under fast loading control, the bottom
portion of the screen will indicate fast loading.
Data collected in Dulbecco’s phosphate buffered
media (no calcium or magnesium) using a 10 µm
carbon-fiber microelectrode. Note the parameter
set installed is indicated in the upper left corner of
the screen (CV01). Effective scan rate 470 V/s.
When the measurement is made in the presence of
10 µM dopamine, the signal is observed as a signal
on top of the background current. In the next note,
analysis of this data will be performed.
Appearance of Clampex after data collection is
complete. Data collected in Dulbecco’s phosphate
buffered media (no calcium or magnesium) using a
10 µm carbon-fiber microelectrode in the presence
of 10 µM dopamine. Parameter set: CV02.
Application Note #4
December 1994
Printed in U.S.A.
Page 4 of 4