BLM KIT
Elements Miniaturized Educational Kit with embedded eONE Amplifier
USER’S QUICK GUIDE
May 2017
elements
enabling technologies for Life Science
BLM EDU KIT guide
This quick guide is intended to give users some brief information on how to
set up a complete Bilayer Lipid Membrane setup using the BLM Kit.
The guide is composed of the following sections:
1) Instructions for software installation
2) BLM Kit content and setup
3) Instructions to properly perform a typical experiment:
1. Electrodes Chlorination
2. BLM formation and protein insertion
3. Teflon septum and BLM chamber’s cleaning
4) Example data obtained from users with the BLM Kit
iii
1) Instructions for software installation
1) Download EDR software from http://elements-ic.com/downloads/
2) Unzip the archive and start the installation
3) Plug the eONE and wait for hardware driver installation
BLM EDU KIT guide
4
2) BLM KIT content and setup
The kit is contained into a 16 x 10 x 5 cm Faraday cage and includes:
• a BLM chamber made by two 1 ml Delrin cuvette with electrode input;
• a set additional laser drilled Teflon septa with fixed hole size of 50, 80, 110 µm;
• a paintbrush,
• Ag electrodes and input connector to plug the eONE amplifier.
BLM
Chamber
Ag/AgCl
electrode
connector
Additional Teflon septa
(with hole sizes of 50,
80 and 110 m)
paintbrush
Figure 1: BLM EDU Kit content
Figure 2: BLM Kit experimental setup example
Technical support & repairs
The typical connection scheme is the following:
Figure 3: BLM Kit connections
Warning:
Do not short-circuit the electrodes with the Faraday cage!
5
BLM EDU KIT guide
6
3) Instructions to properly perform
a typical experiment
3.1) Instructions for Chloriding Ag/AgCl electrodes
Figure 4: Ag/AgCl electrodes connector
1) Clean Ag wires before chloriding it:
o If it is the first usage of the wire, simply clean with ethanol and rinse with
double-distilled water;
o If the wire was previously chlorided, remove the old Ag/AgCl coating by
abrading the surface with an extra fine grit sandpaper, then rinse with ethanol and double-distilled water.
2) Chloride silver wires:
o The simplest method to chloride a silver wire is to immerse it in bleach until
the wire becomes light grey color (typically 15 to 30 minutes are enough).
o Another method that requires a bit more effort but yields a deeper and more
uniform
coating
is
to
use
electroplating.
Chloriding a silver wire using this method is achieved by making it positive
relative to a solution containing NaCl (0.9%) or KCI (1 M) and passing a cur2
rent at a rate of approximately 1 mA/cm for about a minute, or until the wire
is adequately plated and becomes gray color. For example, to chloride a 1
cm length of a 0.5 mm Ag wire (that is the diameter of the wires used in the
kit) requires 0.15 mA of current.
While plating, occasionally reversing the polarity for several seconds tends to
deepen the chloride coating and yield a more stable electrode.
It is suggested to perform at least the first method every day’s work to have stable electrodes.
Technical support & repairs
7
3.2) Instructions for Bilayer Lipid Membrane formation (by painting method)
1) Prepare Lipid solution: 10 mg/ml DPhPC in nonane or decane
(suggested lipids: 4ME 16:0 PC - 1,2-diphytanoyl-sn-glycero-3-phosphocholine,
850356P Avanti Polar Lipids product code).
2) Prepare Buffer solution, for instance 1M KCl, 10mM HEPES PH7 (if needed see
https://www.youtube.com/watch?v=FCBY8c-m04g as an example on how to prepare it).
3) Pretreat the microhole in the Teflon septum using 10% hexadecane in pentane (or
hexane)
solution
to
increase
lipids
affinity
to
the
septum
(Although the pretreatment provides a beneficial effect, it is not essential.)
4) Wait 5 minutes.
5) Start the EDR software,
6) Set the 20nA range;
7) Apply a triangular wave input signal (protocol 1);
8) Fill the BLM chambers with buffer solution above the hole in the Teflon septum
(usually 700 l are enough);
9) Compensate electrode offsets: in the Controls tab, click on Digital Compensation.
The mean current will reach a value close to 0 nA. At this point, uncheck the Digital
Compensation. This action is required to set to 0 the current when a conductive
path is present between the two electrodes, without any bilayer formed or any occlusion in the Teflon septum.
Figure 5: Digital compensation
BLM EDU KIT guide
8
10) In the first usage of a new Teflon septum, the hole can be occluded by air bubbles
resulting in a reduced amplitude square wave similar to the one that can be seen
when BLM is formed; in this case remove the buffer solution and fill the BLM
chamber again increasing the pressure on the hole in the Teflon septum with the
pipette. Another method is to give some mechanical shocks to the chamber in order to fill the hole.
11) Once the triangular wave current signal is present, start to gently paint the lipid solution on the hole in the Teflon septum using the paintbrush;
Figure 6: Painting method
Technical support & repairs
9
12) To monitor membrane formation using the triangular wave input signal:
o When no membrane is present, a full
scale square wave due to amplifier saturation (in case of highly conductive
buffer solution used) or a triangular current signal (in case of low conductive
buffer solution and small holes in Teflon
septum is used) can be seen,
o when membrane is formed a timedependent square wave current signal
can be seen in the display window (its
amplitude increases since it is proportional to BLM capacitance).
Figure 7: No-BLM situation current signal
Figure 8: Formed BLM situation current signal
13) If no membrane is formed, continue to add small quantities of lipids to the septum
using the paintbrush:
o Clean the paintbrush dipping it in a test tube containing ethanol;
o Accurately dry the paintbrush using a wipe;
o Add other lipid solution to the hole in the Teflon septum by painting;
o Repeat the procedure until the membrane is formed.
14) Once the membrane is formed, apply the desired input signal (see the different
voltage protocols in the EDR guide) and add the protein under study.
For instance -hemolysin toxin (aHL - H9395 SIGMA-ALDRICH product code) in
double distilled water (or buffer) solution can be used. It could be useful to prepare
aliquots (e.g. 10 g/ml concentrated aliquots and add something like 0,1-0,5 l to
the chambers, to have a final concentration in the chambers of some ng/ml).
BLM EDU KIT guide
10
Notes to have successful experiments:






Use clean double distilled water and fresh KCl buffer solution.
To avoid "lipid plugs" the lipid solution can be more diluted (like 5 mg/ml DPhPC in
nonane or decane).
After having monitored the BLM formation using the triangular wave protocol, set a
constant voltage (for instance +100 mV). If you can't see any insertion try to add
some more toxin in solution, however the most important thing is the quality of the
BLM. Sometimes it could be useful to break and form the lipid membrane again to
promote BLM thinning and protein insertion and avoid "lipid plug".
A good BLM should have a capacitance of about 25pF or more if you are using Teflon membranes with 80m hole; and a capacitance of at least 35-40 pF if you are
using holes of 110m in diameter.
If capacitance is lower of these values for a long time (15-20 min) it is probably a
“lipid plug”.
To avoid lipid plugs also try to dry with a wipe as better as possible the paintbrush
before dipping it in the lipid solution .
It is important you use fresh aHL aliquot (if aHL aliquots are used), not frozen more
than one time.
Since the aHL protein is very slightly negatively charged in buffer solution at pH 7, it
could be useful to add the toxin in the cis chamber (at ground) if constant positive
voltages are then applied.
Technical support & repairs
11
3.3) Instructions for Teflon septum and BLM chamber’s cleaning
Warning:
Do not use Acetone to clean Teflon septum
o The simplest way is to clean the Teflon septum by using ethanol to remove lipids, then
simply rinse the septum with double-distilled water.
o Another procedure is to use trisodium phosphate (TSP, Na3PO4) solution (40-50 mM),
which is a stronger cleaner than ethanol.
In this case you can use the following procedure to clean the supports:
1) Rinse the chambers with double-distilled water to remove your chemicals and biological;
2) Remove the Teflon support from the BLM chamber
3) Clean the hole in the septum using the trisodium phosphate (TSP) solution;
4) Rinse with dilute HCl solution (0.1% by volume) to remove any residual TPS;
5) Rinse with double-distilled water to remove any remaining HCl;
6) Gently dry the septum using a wipe or a low air jet.
Warning:
Since the Teflon septum is very fragile, be careful to not apply a high pressure on them while rinsing or drying.
Figure 9: BLM chamber and Teflon support (with a 80m diameter hole)
BLM EDU KIT guide
12
4) Example data obtained from users
with the BLM Kit
of PEG 1500 through a -hemolysin nanopore.
Buffer solution: 3M KCl. -40mV transmembrane voltage applied. Data were recorded at 10 KHz final bandwidth (20 KHz sampling rate without filter).
(Data from A. Oukhaled, LAMBE UMR 8587 CNRS, Évry and Cergy University,
Cergy-Pontoise 95011 cedex, France)
Figure 10: Translocations
1
Current pulses of PEG 4000 through single -hemolysin nanopore.
Buffer solution: 4M KCl. -40 mV transmembrane voltage applied. Data recorded at
10 KHz final bandwidth (20 KHz sampling rate without filter).
Figure 11:
Current pulses of Poly(dC)50 through single -hemolysin nanopore.
Buffer solution: 1 M KCl. 100 mV transmembrane voltage applied. Data recorded at
5 KHz final bandwidth (10 KHz sampling rate without filter).
Figure 12:
(Data from Prof. Zhi-Yong Wu’s group, Research Center for Analytical Sciences,
College of Sciences, Northeastern University, Shenyang, Liaoning 110819, China)
Technical support & repairs
13
Technical support & repairs
ELEMENTS S.R.L. offers wide-ranging, complete after-sales technical support.
The staff who deal with this handle questions on the entire range of products skillfully, quickly, and efficiently.
You can send an e-mail or phone our staff in the service department, and they will give you complete, prompt advice
on how to resolve your problems.
Elements s.r.l.
Viale G. Marconi, 438
47521 Cesena
ITALY
tel: +39 0547 482983
email: [email protected]
web: www.elements-ic.com
Index of figures
Figure 1: BLM EDU Kit content ........................................................................................................................................ 4
Figure 2: BLM Kit experimental setup example ................................................................................................................. 4
Figure 3: BLM Kit connections .......................................................................................................................................... 5
Figure 4: Ag/AgCl electrodes connector ............................................................................................................................ 6
Figure 5: Digital compensation .......................................................................................................................................... 7
Figure 6: Painting method .................................................................................................................................................. 8
Figure 7: No-BLM situation current signal ........................................................................................................................ 9
Figure 8: Formed BLM situation current signal ................................................................................................................ 9
Figure 9: BLM chamber and Teflon support (with a 80 m diameter hole) ..................................................................... 11
Figure 10: Translocations of PEG 1500 through a -hemolysin nanopore. ................................................................... 12
Figure 11: Current pulses of PEG 4000 through single -hemolysin nanopore. ............................................................ 12
Figure 12: Current pulses of Poly(dC)50 through single -hemolysin nanopore. ........................................................... 12
elements
enabling technologies for Life Science
Elements s.r.l.
Viale G. Marconi, 438
47521 Cesena
ITALY
tel: +39 0547 482983
email: [email protected]
web: www.elements-ic.com