Alpha-helical peptide-induced vesicle rupture revealing new insight into the vesicle fusion process as monitored in situ by quartz crystal microbalance-dissipation and reflectometry.

We have used simultaneous quartz crystal microbalance-dissipation (QCM-D) monitoring and four-detector optical reflectometry to monitor in situ the structural transformation of intact vesicles to a lipid bilayer on a gold surface. The structural transformation of lipid vesicles to a bilayer was achieved by introducing a particular amphipathic, alpha-helical (AH) peptide. The combined experimental apparatus allows us to simultaneously follow the acoustic and optical property changes of the vesicle rupturing process upon interaction with AH peptides. While QCM-D and reflectometry have similar sensitivities in terms of mass and thickness resolution, there are unique advantages in operating these techniques simultaneously on the same substrate. These advantages permit us to (1) follow the complex interaction between AH peptides and intact vesicles with both acoustic and optical mass measurements, (2) calculate the amount of dynamically coupled water during the interaction between AH peptides and intact vesicles, (3) demonstrate that the unexpectedly large increase of both adsorbed mass and the film's energy dissipation is mainly caused by swelling of the vesicles during the binding interaction with AH peptides, and (4) permit us to understand the structural transformation from intact vesicles to a bilayer via the AH peptide interaction by monitoring viscoelastic properties, acoustic mass, optical mass, and thickness changes of both the binding and destabilization processes. From the deduced "hydration signature" we followed the complex transformation of lipid assemblies. On the basis of this information, a mechanism of this structural transformation is proposed that provides new insight into the process of vesicle fusion on solid substrates.

QCM-D and reflectometry instrument: applications to supported lipid structures and their biomolecular interactions.

A novel setup was recently developed, combining quartz crystal microbalance with dissipation monitoring (QCM-D) and optical reflectometry for measurements on one and the same surface of, for example, biomolecular adlayers and interactions ( Rev. Sci. Instr. 2008 , 79 075107 ). This combination was chosen on the basis of prior experience of using QCM-D and optical techniques in separate instruments, which showed both the advantage of employing multiple techniques and the disadvantage of not working with the same surface and (flow) cell. The new instrument provides, for example, information about associated water and structural changes of the adlayers that would often pass unnoticed or be hard to interpret or quantify, using either technique alone. The triple response instrument (QCM-D frequency and dissipation and reflectometry) is here applied to four model systems: (A) formation of supported lipid bilayers (SLBs), (B) lipid exchange between a SLB and transiently adsorbed vesicles, (C) binding of a hydrated peptide on a functionalized SLB, and (D) streptavidin coupling to a biotinylated SLB, followed by attachment of biotinylated vesicles. The results demonstrate three major advantages of the combination instrument: (i) much faster data collection because the experiments are done on one surface for all signals, (ii) a common time axis and the same relative importance of surface kinetics and mass transport because the same liquid sample and the same transport conditions apply, and (iii) new features are discovered about the studied system that would be difficult to unravel in separate instruments.

A combined nanoplasmonic and electrodeless quartz crystal microbalance setup.

We have developed an instrument combining localized surface plasmon resonance (LSPR) sensing with electrodeless quartz crystal microbalance with dissipation monitoring (QCM-D). The two techniques can be run simultaneously, on the same sensor surface, and with the same time resolution and sensitivity as for the individual techniques. The electrodeless QCM eliminates the need to fabricate electrodes on the quartz crystal and gives a large flexibility in choosing the surface structure and coating for both QCM-D and LSPR. The performance is demonstrated for liquid phase measurements of lipid bilayer formation and biorecognition events, and for gas phase measurements of hydrogen uptake/release by palladium nanoparticles. Advantages of using the combined equipment for biomolecular adsorption studies include synchronized information about structural transformations and extraction of molecular (dry) mass and degree of hydration of the adlayer, which cannot be obtained with the individual techniques. In hydrogen storage studies the combined equipment, allows for synchronized measurements of uptake/release kinetics and quantification of stored hydrogen amounts in nanoparticles and films at practically interesting hydrogen pressures and temperatures.

A combined reflectometry and quartz crystal microbalance with dissipation setup for surface interaction studies.

We have developed an instrument for surface interaction studies, which combines a newly invented four detector optical reflectometry setup with quartz crystal microbalance with dissipation (QCM-D) monitoring. The design is such that data from both techniques can be obtained simultaneously on the same sensor surface, with the same signal-to-noise ratio and time resolution, as for the individual techniques. In addition, synchronized information about structural transformations, molecular mass, and the hydration of thin films on solid surfaces can be obtained on the same specimen, as validated by monitoring the formation of supported lipid bilayers on a silica-coated QCM sensor surface. We emphasize that the optical (molecular) mass can be separated from the acoustic mass including hydrodynamically coupled solvent, which means, in turn, that the amount of solvent sensed by the QCM-D technique can be dynamically resolved during adsorption processes. In addition, the advantage/necessity to use four, compared to two, detector reflectometry is emphasized.

Investigation of binding event perturbations caused by elevated QCM-D oscillation amplitude.

We report measurements with the quartz crystal microbalance with dissipation monitoring (QCM-D) technique, with focus on how the shear oscillation amplitude of the sensor surface influences biorecognition binding events. Technically, this is made as reported recently (M. Edvardsson, M. Rodahl, B. Kasemo, F. Höök, Anal. Chem., 2005, 77(15), 4918-4926) by operating the QCM in dual frequency mode; one harmonic (n = n1) is utilized for continuous excitation of the QCM-D sensor at resonance at variable driving amplitudes (1-10 V), while the second harmonic (n not equaln(1)) is used for combined f and D measurements. By using one harmonic as a "probe" and the other one as an "actuator", elevated amplitudes can be used to perturb - or activate - binding reactions in a controlled way, while simultaneously maintaining the possibility of probing the adsorption and/or desorption events in a non-perturbative manner using combined f and D measurements. In this work we investigate the influence of oscillation amplitude variations on the binding of NeutrAvidin-modified polystyrene beads (slashed circle approximately 200 nm) to a planar biotin-modified lipid bilayer supported on an SiO2-modified QCM-D sensor. These results are further compared with data on an identical system, except that the NeutrAvidin-biotin recognition was replaced by fully complementary DNA hybridization. Supported by micrographs of the binding pattern, the results demonstrate that there exists, for both systems, a unique critical oscillation amplitude, A(c), below which binding is unaffected by the oscillation, and above which binding is efficiently prevented. Associated with A(c), there is a critical crystal radius, r(c), defining the central part of the crystal where binding is prevented. From QCM-D data, A(c) for the present system was estimated to be approximately 6.5 nm, yielding a value of r(c) of approximately 3 mm--the latter number was nicely confirmed by fluorescent- and dark-field micrographs of the crystal. Furthermore, the fact that A(c) is observed to be identical for the two types of biorecognition reactions suggests that it is neither the strength, nor the number of contact points, that determine the amplitude at which binding is prevented. Rather, particle size seems to be the determining parameter.

Light-regulated release of liposomes from phospholipid membranes via photoresponsive polymer-DNA conjugates.

A method for releasing tethered liposomes from a supported lipid bilayer in response to a light stimulus is described. The tethering is accomplished through the hybridization of end-functionalized DNA that resides on both the supported lipid bilayer and liposome surfaces. Normally consisting of cholesterol or lipid tails, the end group is replaced in this study by a photoresponsive polymer that partitions into lipid bilayers at physiological pH. When exposed to UV light, it undergoes excited state proton transfer with water. The ensuing increase in polarity increases the solubility of the polymer in the aqueous phase. Quartz crystal microbalance with dissipation monitoring (QCM-D) and fluorescence microscopy have been used to record both the construction of the vesicle assembly and the subsequent response to UV light. It is found that the critical flow rate for vesicle release is reduced when buffer flow is performed in conjunction with UV exposure.

A dual-frequency QCM-D setup operating at elevated oscillation amplitudes.

An often raised, but rarely addressed, question with respect to applications of the quartz crystal microbalance technique is whether the shear oscillation of the sensor surface influences the adsorption kinetics or binding events being studied. Motivated by this uncertainty, as well as by the possibility of using elevated amplitudes to influence and steer specific biomolecular interactions, we have further developed the quartz crystal microbalance with dissipation monitoring (QCM-D) technique to operate in dual-frequency mode. One mode (one harmonic) is utilized for continuous excitation of the QCM-D sensor at resonance, at variable driving amplitudes, while the other mode (another harmonic) is used for combined frequency and energy dissipation (damping) measurements. To evaluate this experimental approach, we investigated the following: (i) the well-established process by which intact lipid vesicles adsorb and decompose into a planar supported lipid bilayer on SiO2, recently shown to be very sensitive to external perturbations, and (ii) specific streptavidin binding to biotin-modified surfaces. In the former case, we observed a clear influence of elevated oscillation amplitudes on the bilayer formation kinetics, while in the latter case, no influence was observed for protein monomers. However, binding was inhibited when the biotin-binding protein was coupled to colloidal particles (o.d. approximately 200 nm).