Using Axon Instruments Hardware for Electrochemistry

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Axon Instruments, Inc.
1101 Chess Drive, Foster City, CA 94404, USA
Phone (415) 571-9400 Fax (415) 571-9500
e-mail: [email protected]
Marketing & Scientific Applications
Using Axon Instruments Hardware for Electrochemistry
Overview
This note is a guide for configuring Axon Instruments hardware to make electrochemical measurements. The
primary focus is amperometry at single cells. Switching the configuration of the instrument for voltammetry (or
stepped potential techniques) requires a change in the sensitivity of the headstage to generate the applied voltage
signal. Details on using pCLAMP for voltammetric measurements can be found inApplication Notes 4 and 5.
Many of Axon’s amplifiers are capable of supporting electrochemistry. A list of the instruments and modifications
is outlined in the table below. Modifications are primarily for the purpose of extending the command voltage
range of the instrument.
Instrument
Command
Voltage Range
Modifications
Application
GeneClamp 500
± 2.0 V
None
Voltammetry / Amperometry
Axopatch 200A
± 1.0 V
Procedure #22
Voltammetry / Amperometry
Axopatch-1D
± 0.6 V
Procedure #14
Amperometry
CyberAmp with AI 403
± 1.0 V
This App. note, page 4
Amperometry
Procedure #22 - Axopatch 200/200A Voltammetry Modification
- Configures the instrument for increased voltage range.
Procedure #14 - Axopatch-1A,1B,1C,1D Command Gain Modification
- Increases the voltage command of the 1D to 100 mV/V
(± 6 V maximum analog input).
Most of the modifications described above can be performed in the lab or you can send the instrument to Axon to
have the modification performed by an Axon technician. In addition, for the Axopatch-1D and 200A, you can
request the capacitance dither kit ( Procedure #23) to add calibrated membrane capacitance measurement
capability.
Procedure #23 - Capacitance Dither Kit- Allows calibration of capacitance measurements using AxoBASIC software routines.
Application Note #6
Part Number 9110-001 Rev A
May 1995
Printed in U.S.A.
Page 1 of 4
GeneClamp 500
The GeneClamp 500 can be used almost immediately for electrochemistry measurements. Use the following chart
to select headstages for your particular application. In particular, select the smallest current range which will fit
your needs. This will ensure the best amplifier noise performance for your measurements (please refer to Axon’s
Product Catalog for noise specifications).
GeneClamp 500 Headstages for Electrochemistry
Headstage
Current Range
Example Applications
CV-5-100MU
± 100 nA
Fast scan cyclic voltammetry
CV-5-1GU
± 10 nA
Vesicular measurements at chromaffin cells
CV-5-100GU
± 0.1 nA
Electrochemistry at neurons
Instrument Configuration
The headstage should be connected to the electrode through a stable drift-free interface such as the HL-U electrode
holder. In addition, a stable reference potential is required and is obtained by connecting a reference electrode
through the ground of the headstage. The reference electrode can be placed directly into the bath of the
preparation or can be isolated from the solution. This is done by placing the reference electrode into a buffered
filled micropipette whose tip has been partially blocked with glass wool or Agar (prepared with buffer ions). This,
in effect, helps prevent silver ions from the reference electrode from contaminating the solution. Reference
electrodes can be obtained from Axon Instruments ( HLA−
−003). All connections should be made as short as
possible to reduce noise pickup. Care should be taken to avoid bubbles in agar electrodes, since these also increase
noise.
The CV-5 headstage is connected to the HEADSTAGE #3 (PATCH) input of the instrument. To set the electrode
voltage, select the V3 button to display the voltage on the DC METER panel and dial in the DC COMMAND
POTENTIAL desired (you must select the polarity). If you are using the scaled output, select the I3 button to
monitor the electrode current and select a filter frequency and gain. If you will be using pCLAMP’s Fetchex or
AxoTape to record the current measurements, you will want to set up telegraphing to record the instrument options
selected. The configuration described is suitable for making amperometric measurements at single cells. For
voltammetry, you will have to provide an input command voltage through the External Patch Clamp Command
input on the rear panel. If you will be making voltammetric measurements, you should refer to Application Notes
4 and 5.
Application Note #6
Part Number 9110-001 Rev A
May 1995
Printed in U.S.A.
Page 2 of 4
Axopatch 200A
The Axopatch 200A is an ideal instrument for the electrochemical measurement of quantal release. The Axopatch
200A is a good instrument for voltammetry (in whole cell mode), but the strength of the instrument is in its lownoise patch amplifier circuitry. The Axopatch 200A contributes only 0.02 pA rms (0.1-1 kHz) to the overall noise
while providing a large dynamic range. The best electrochemical sensors used for these studies have an associated
capacitance of 2-10 pF which yields about 1 pA p-p of noise 0.03-1 kHz. Unlike many amplifiers (based on resistive
feedback technology), the 200A will allow you to maximize the advantage of using low-noise electrodes.
Modifications
If your electrochemical measurements require a voltage range > 200 mV, you must perform the modifications
described in Procedure #22 or have a suitable source for overdriving the voltage command input.
Do not
overdrive an Axopatch 200; this will damage the instrument. There are several ways to obtain the ±1 V range:
change the holding potential to 100 mV/turn, or change the external command sensitivity (front or rear switched)
to 100 mV/V.
The field kit for the modification contains the required resistors, labels and instructions and is provided by Axon
free of charge. If you choose not to modify your instrument, command voltages >200 mV can be obtained by
driving the command voltage input to voltages greater than 10 V (overdriving). For example, to get 700 mV of
applied potential, 35 V will have to be applied to the external command input (20 mV/V scaling). The amount of
external voltage command required can be reduced if the holding command potential is used simultaneously. The
voltage applied to the electrode is limited to about 1.1 V.
Instrument Configuration
The instrument MODE should be in V-CLAMP, and you should use either a CV 201AU or CV 202AU headstage.
Working and reference electrode ( e.g., carbon-fiber electrode and silver-silver chloride electrode) connections are
similar to those described above for the CV-5 headstages. Even with the voltage command modification, the panel
METER will read a maximal value of ±200 mV. Consequently, you should monitor the rear panel connector
labeled 10 Vm while adjusting the HOLDING COMMAND knob (the polarity must be chosen). Alternatively,
you can set the potential using one of the external voltage command inputs. The amplifier CONFIG switch should
be in PATCH MODE, and the gain will have to be set according to the number and size of quanta observed in
your preparation. In addition, for controlled stimulus experiments, the integration can be reset manually at the
appropriate time by applying a TTL pulse to the FORCED RESET INPUT on the rear panel. Series resistance
compensation should be turned off.
Axopatch-1D
The Axopatch-1D is also a good option for electrochemistry experiments. Internally, however, the voltage applied
to the electrode is limited to about 630-650 mV, which would preclude its use with some voltammetry protocols.
Modifications
Modifications to the Axopatch-1D are required only when the external command voltages you desire are greater
than 200 mV and cannot be obtained by overdriving the external command input. Modification of the command
input range for the Axopatch-1D is covered in Procedure #14. The command voltage range of the Axopatch-1D
cannot be increased to greater than 630-650 mV.
Instrument Configuration
Set β=1 and the instrument MODE as V-CLAMP. Working and reference electrode ( e.g., carbon-fiber electrode
and silver−silver chloride electrode) connections are similar to those described above for the CV-5 headstages. The
options for obtaining 600 mV from the Axopatch-1D are given in the table below.
Ext. Command
30 V (20 mV/V)
10 V
6 V (Procedure #14)
*
*
Axopatch-1D settings for Amperometry at 600 mV
Step Command
Holding Potential
0 mV
0 mV
199.9 mV (Continuous)
200 mV
0 mV
0 mV
Other options should be turned off (speed test, seal test, etc.). Series resistance and capacitance compensation are not required.
The gain of the amplifier should be set to β=1 for large signals ( ±20 nA) and β=100 for small signals ( ±200 pA).
System noise will be best forβ=100 (0.04 pArms, DC-1 kHz bandwidth).
Application Note #6
Part Number 9110-001 Rev A
May 1995
Printed in U.S.A.
Page 3 of 4
CyberAmp/AI 403 SmartProbe Combination
The CyberAmp’s AI 403 SmartProbe amplifier is a convenient solution for measuring quantal secretion from
single cells. The AI 403 picoammeter has approximately a ±1 nA range. The AI 403 does not have a built-in
command voltage; however, this can be overcome by using a small voltage offset circuit. The primary goal of the
voltage offset circuit is to provide a voltage difference between the working and reference electrodes. For the case
of catecholamines, a 600-800 mV voltage is sufficient to result in quantitative oxidation. This can be done either
by setting the reference at -800 mV and leaving the working electrode at virtual ground or by leaving the reference
electrode at virtual ground and offsetting the electrode potential by+800 mV.
Modifications: Voltage Offset Circuit
The diagram below describes in greater detail the type of circuit that can be constructed to provide an
800 mV offset. Since noise on the potential source can adversely affect the current signal, the input voltage is
divided by approximately 10/1 to reduce noise. A standard 10-turn 100 k Ω potentiometer (pot) is used. The
supply voltage is applied to the end terminals of the potentiometer and the negative terminal. The potential of the
center arm (wiper) of the pot is adjusted to the desired voltage ( e.g., 800 mV). The reference electrode is
connected to the ground terminal and the potential is then applied across the ground and negative terminals of the
AI 403 input. By interposing the potential between the reference (ground) and working electrodes, the bath
potential remains at system ground and the working electrode is driven to the command voltage. If you do not
have a convenient low-noise 10 V (DC) power supply, a battery can also be used; however, the pot should be
adjusted to reflect the new power supply.
Monitoring the Current
The current is monitored on the output BNC of the AI 403 channel of the CyberAmp. Since there is a voltage
offset in the current preamplifier, there will be a corresponding current offset in the output. This value will vary
according to the command potential you have selected. Record this value as the zero current reference.
AI 403 Command Voltage Circuit
In this configuration of the AI 403 command voltage circuit, the reference electrode will be connected to the
ground (yellow jack) of the AI 403 amplifier. The working electrode is connected to the Input (red) connector via
an external clip ( e.g., "Alligator clip"). The length between the connection to the electrode and the amplifier
should be kept to a minimum to reduce noise pickup.
Application Note #6
Part Number 9110-001 Rev A
May 1995
Printed in U.S.A.
Page 4 of 4
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