further extrusion steps or a longer period of sonication can be
performed. Potentially, extrusion could also be performed with
a 30-nm-pore polycarbonate membrane, which we have found
to produce vesicles of approximately 50–60 nm. Lipid suspension may also benefit from freeze-thawing (4Â cycles of placing
the sample in liquid nitrogen and then 50
C water bath)
posthydration and prior to extrusion/sonication.
11. If cleaned and stored correctly, SiO 2 Qsense sensors can be
reused 10–30 times.
12. We would recommend using reverse action tweezers. The
tweezers should have a round end and smooth gripping surfaces to avoid damage to sensors. Hold the sensors from
underneath to prevent washing contaminants from the tweezers onto the sensor surface.
13. Liquid may remain on the edge of the sensor under the tweezers. This can be wicked off with a clean, lint-free tissue.
14. Sensors should be used for lipid deposition immediately after
UV Ozone cleaning. This is due to the preparation step
increasing the charge on the sensor surface. Sensors can be
stored dry at the post ethanol clean step but would require
ethanol rinse and nitrogen dry again to ensure no dust on the
surface. Proceed with UV Ozone clean.
15. If liquid is present in the chamber void or under the O-ring,
this can be dried with a lint-free tissue.
16. If alternative lipids are to be used, then the temperature needs
to be set above the lipid T m . Once bilayer has formed, the
temperature can be returned to 20
C; however, this will affect
the fluidity of the formed bilayer.
17. To avoid disturbances in f and D, it is vital to use the same stock
of 10 mM HEPES, 150 mM NaCl, and 2 mM CaCl 2 (pH 7.4)
for running buffer and all subsequent sample dilution steps.
18. Drift should be around 2 Hz/h ( f ) and 0.2 Â 10
À6 (D). Noise
levels should be lower than 0.6 Hz (peak-to-peak) and
0.15 Â 10
À6 (peak-to-peak), respectively, when measured
within a period of 2 min. If the sensor is failing to equilibrate,
this could indicate dirty tubing, unfiltered/old buffer, or a
dirty sensor. Buffer should be made fresh. System can be
flushed with 2% SDS, ultrapure water, ethanol, and a final
rinse with ultrapure water. Repeat cleaning and preparation
procedure for Qsense sensors. If following these steps the
signal still fails to stabilize during equilibration, then the sensor
may be faulty and will need to be replaced. Alternatively, the
flow module may require cleaning, which can be performed as
per the manufacturer’s instructions.
194
Holly L. Birchenough and Thomas A. Jowitt
performed. Potentially, extrusion could also be performed with
a 30-nm-pore polycarbonate membrane, which we have found
to produce vesicles of approximately 50–60 nm. Lipid suspension may also benefit from freeze-thawing (4Â cycles of placing
the sample in liquid nitrogen and then 50
C water bath)
posthydration and prior to extrusion/sonication.
11. If cleaned and stored correctly, SiO 2 Qsense sensors can be
reused 10–30 times.
12. We would recommend using reverse action tweezers. The
tweezers should have a round end and smooth gripping surfaces to avoid damage to sensors. Hold the sensors from
underneath to prevent washing contaminants from the tweezers onto the sensor surface.
13. Liquid may remain on the edge of the sensor under the tweezers. This can be wicked off with a clean, lint-free tissue.
14. Sensors should be used for lipid deposition immediately after
UV Ozone cleaning. This is due to the preparation step
increasing the charge on the sensor surface. Sensors can be
stored dry at the post ethanol clean step but would require
ethanol rinse and nitrogen dry again to ensure no dust on the
surface. Proceed with UV Ozone clean.
15. If liquid is present in the chamber void or under the O-ring,
this can be dried with a lint-free tissue.
16. If alternative lipids are to be used, then the temperature needs
to be set above the lipid T m . Once bilayer has formed, the
temperature can be returned to 20
C; however, this will affect
the fluidity of the formed bilayer.
17. To avoid disturbances in f and D, it is vital to use the same stock
of 10 mM HEPES, 150 mM NaCl, and 2 mM CaCl 2 (pH 7.4)
for running buffer and all subsequent sample dilution steps.
18. Drift should be around 2 Hz/h ( f ) and 0.2 Â 10
À6 (D). Noise
levels should be lower than 0.6 Hz (peak-to-peak) and
0.15 Â 10
À6 (peak-to-peak), respectively, when measured
within a period of 2 min. If the sensor is failing to equilibrate,
this could indicate dirty tubing, unfiltered/old buffer, or a
dirty sensor. Buffer should be made fresh. System can be
flushed with 2% SDS, ultrapure water, ethanol, and a final
rinse with ultrapure water. Repeat cleaning and preparation
procedure for Qsense sensors. If following these steps the
signal still fails to stabilize during equilibration, then the sensor
may be faulty and will need to be replaced. Alternatively, the
flow module may require cleaning, which can be performed as
per the manufacturer’s instructions.
194
Holly L. Birchenough and Thomas A. Jowitt