19. If lipid deposition does not show the responses demonstrated
here, this indicates that a full lipid bilayer may not have been
formed. A large decrease in f (and high D), which plateaus,
indicates intact liposomes that have not popped to form a
bilayer. This is due to liposomes that are too large, forgetting
to add calcium or dirty sensor surfaces. Further extrusion or
sonication would be required (see Note 10). It is also possible
that the surface charge has been lost and so the sensor cleaning
and preparation steps should be repeated. A decrease in f to
approximately À25 Hz without the dip indicates that the vesicles are popping as soon as they reach the surface and no
liposome critical coverage has been achieved before popping.
This can give incomplete bilayers and is due to vesicles that are
too small. Unilamellar vesicle preparation should be repeated.
If the return in frequency to À25 Hz is slow (inverse peak is
shifted to the right) and D signal is high, this could indicate
that the lipid layer contains some vesicles that have not popped.
This is due to a nonhomogeneous vesicle preparation that
includes larger liposomes. Further extrusion or sonication
would be required (see Note 10).
20. As a measure to check completeness of layer coverage and
passivation, 50 μg/mL BSA in running buffer can be flowed
over the surface at 50 μL/min for 10 min. A passivated layer
should not show changes in f or D.
21. Once bilayer has formed, the running buffer can be changed at
this point, e.g., to exclude CaCl 2 . A change in running buffer
will cause a change in f and D and so a period of equilibration
will be required to achieve a stable signal again. If proceeding
with the maleimide linker protocol, then ensure that running
buffer is sulfhydryl-free.
22. It is possible to control the level of biotinylated molecule to be
subsequently immobilized to the surface by controlling the
amount of avidin/streptavidin immobilized to the lipid surface. This can be achieved by reducing the time and/or concentration of avidin/streptavidin introduced to the surface.
Acknowledgments
Figures were produced using BioRender with thanks to Maryline
Fresquet and The Wellcome Trust for funding.
QCM-D Sensor Preparation to Study Complex Interactions
195
here, this indicates that a full lipid bilayer may not have been
formed. A large decrease in f (and high D), which plateaus,
indicates intact liposomes that have not popped to form a
bilayer. This is due to liposomes that are too large, forgetting
to add calcium or dirty sensor surfaces. Further extrusion or
sonication would be required (see Note 10). It is also possible
that the surface charge has been lost and so the sensor cleaning
and preparation steps should be repeated. A decrease in f to
approximately À25 Hz without the dip indicates that the vesicles are popping as soon as they reach the surface and no
liposome critical coverage has been achieved before popping.
This can give incomplete bilayers and is due to vesicles that are
too small. Unilamellar vesicle preparation should be repeated.
If the return in frequency to À25 Hz is slow (inverse peak is
shifted to the right) and D signal is high, this could indicate
that the lipid layer contains some vesicles that have not popped.
This is due to a nonhomogeneous vesicle preparation that
includes larger liposomes. Further extrusion or sonication
would be required (see Note 10).
20. As a measure to check completeness of layer coverage and
passivation, 50 μg/mL BSA in running buffer can be flowed
over the surface at 50 μL/min for 10 min. A passivated layer
should not show changes in f or D.
21. Once bilayer has formed, the running buffer can be changed at
this point, e.g., to exclude CaCl 2 . A change in running buffer
will cause a change in f and D and so a period of equilibration
will be required to achieve a stable signal again. If proceeding
with the maleimide linker protocol, then ensure that running
buffer is sulfhydryl-free.
22. It is possible to control the level of biotinylated molecule to be
subsequently immobilized to the surface by controlling the
amount of avidin/streptavidin immobilized to the lipid surface. This can be achieved by reducing the time and/or concentration of avidin/streptavidin introduced to the surface.
Acknowledgments
Figures were produced using BioRender with thanks to Maryline
Fresquet and The Wellcome Trust for funding.
QCM-D Sensor Preparation to Study Complex Interactions
195