that can be used to attach molecules. These can be, for example,
biotin for avidin attachment (and thus biotinylated-protein attachment) or nitrilotriacetic acid (NTA) for the attachment of
hexahistidine-labeled proteins. These layers can then be used for
further protein–ligand interaction analysis. Lipid layers, as well as
being adaptable, also have the potential to deliver a surface that is
less chemically reactive, therefore reducing nonspecific background
binding.
Quartz crystal microbalance with dissipation monitoring
(QCM-D) is a biosensor technique well suited to the analysis of
lipid bilayer deposition and the subsequent analysis of complex
interactions. It is a mass sensing technique also capable of measuring the physical properties of molecules/complexes associated with
the sensor surface [15, 16]. Sensors consist of a thin quartz crystal
disc, often coated with SiO 2 , sandwiched between a pair of electrodes. Application of a voltage via the electrodes to the quartz causes
oscillation of the crystal at a specific resonant frequency. Addition of
mass at the sensor surface leads to a detectable decrease in the
oscillation frequency ( f ) of the crystal producing measurements
of mass change in real time. If the layer produced by mass addition
at the sensor surface is thin and rigid, then the decrease in f is
proportional to the mass of the layer. In most instances, certainly
with biological samples, addition of mass at the sensor surface will
also include coupled water, and analysis of the “softness” of layers
produced provides information on their structural properties.
These structural aspects can be measured simultaneously to mass
changes by measuring the dampening or dissipation (D) of the
oscillation of the crystal. “Soft” or viscoelastic layers increase the
dampening of the signal, resulting in an increase in dissipation
readings.
QCM-D is particularly amenable for the study of lipid bilayer
formation as it is able to detect the addition and loss of water during
bilayer deposition [17–19]. The stages of bilayer formation on solid
supports have been well characterized and are illustrated in Fig. 1,
which demonstrates the multistage nature of lipid deposition [17–
20]. Formation of lipid bilayers on QCM-D SiO 2 sensors follows
the method pioneered by McConnell [21] and has been extensively
studied previously; therefore, a well-established protocol for production is available [17, 18, 22]. With the incorporation of functional lipids in the lipid bilayer, further molecules can be attached.
In this way, layers of molecules can be added to the surface so that
complex interactions can be investigated. Indeed, the surfaces are
large enough that the sensors can be removed and the protein/lipid
layer can be extracted for western blot or silver stain analysis, which
provides invaluable information about what is left on the surface
after complex interactions have taken place.
184
Holly L. Birchenough and Thomas A. Jowitt
biotin for avidin attachment (and thus biotinylated-protein attachment) or nitrilotriacetic acid (NTA) for the attachment of
hexahistidine-labeled proteins. These layers can then be used for
further protein–ligand interaction analysis. Lipid layers, as well as
being adaptable, also have the potential to deliver a surface that is
less chemically reactive, therefore reducing nonspecific background
binding.
Quartz crystal microbalance with dissipation monitoring
(QCM-D) is a biosensor technique well suited to the analysis of
lipid bilayer deposition and the subsequent analysis of complex
interactions. It is a mass sensing technique also capable of measuring the physical properties of molecules/complexes associated with
the sensor surface [15, 16]. Sensors consist of a thin quartz crystal
disc, often coated with SiO 2 , sandwiched between a pair of electrodes. Application of a voltage via the electrodes to the quartz causes
oscillation of the crystal at a specific resonant frequency. Addition of
mass at the sensor surface leads to a detectable decrease in the
oscillation frequency ( f ) of the crystal producing measurements
of mass change in real time. If the layer produced by mass addition
at the sensor surface is thin and rigid, then the decrease in f is
proportional to the mass of the layer. In most instances, certainly
with biological samples, addition of mass at the sensor surface will
also include coupled water, and analysis of the “softness” of layers
produced provides information on their structural properties.
These structural aspects can be measured simultaneously to mass
changes by measuring the dampening or dissipation (D) of the
oscillation of the crystal. “Soft” or viscoelastic layers increase the
dampening of the signal, resulting in an increase in dissipation
readings.
QCM-D is particularly amenable for the study of lipid bilayer
formation as it is able to detect the addition and loss of water during
bilayer deposition [17–19]. The stages of bilayer formation on solid
supports have been well characterized and are illustrated in Fig. 1,
which demonstrates the multistage nature of lipid deposition [17–
20]. Formation of lipid bilayers on QCM-D SiO 2 sensors follows
the method pioneered by McConnell [21] and has been extensively
studied previously; therefore, a well-established protocol for production is available [17, 18, 22]. With the incorporation of functional lipids in the lipid bilayer, further molecules can be attached.
In this way, layers of molecules can be added to the surface so that
complex interactions can be investigated. Indeed, the surfaces are
large enough that the sensors can be removed and the protein/lipid
layer can be extracted for western blot or silver stain analysis, which
provides invaluable information about what is left on the surface
after complex interactions have taken place.
184
Holly L. Birchenough and Thomas A. Jowitt