Probing tethered vesicle assemblies using quartz crystal microbalance with dissipation monitoring: Antibody binding and other applications towards ex vivo, label-free membrane protein analysis.

摘要

Implementing the molecular recognition properties of membrane proteins for applications such as biosensors, diagnostic arrays, and "lab-on-a-chip" devices requires the construction of a model membrane platform able to mimic the unique environment of biological membranes. Using quartz crystal microbalance with dissipation monitoring (QCM-D), fluorescence microscopy, and dynamic light scattering (DLS), this work quantitatively explores a tethered vesicle platform in which phospholipid vesicles are tethered to a planar supported lipid bilayer through biotin-streptavidin linkages. By housing membrane proteins in the vesicle, such an assembly can potentially overcome deleterious protein-substrate interactions while providing a robust, fluid, defect-free model membrane able to house a variety of functional integral proteins. The assembly also permits lateral mobility of the tethered vesicles as well as the use of QCM-D, a powerful surface-sensitive technique, to investigate binding to membrane components in a realtime, label-free fashion.; Each step in the sequential, self assembly-based construction process was characterized quantitatively. Further, antibody binding to an antigen-functionalized tethered vesicle assembly was quantified through viscoelastic modeling of QCM-D measurements and independently through the use of ELISA assays. This provided valuable insights in the modeling and interpretation of QCM-D responses for biomolecular interactions with tethered vesicles, which exhibit significant viscoelastic character.; A direct, rupture-on-contact pathway was demonstrated for lipid bilayer formation from osmotically shocked lipid vesicles. The subsequent adsorption and binding of streptavidin, vesicles, and streptavidin-coated microspheres revealed that there exists a critical surface density of a given species above which interstitial water contributes significantly to the response of the quartz resonator in a phenomenon similar to that produced by surface roughness. Treating water as an additional inertial mass, Sauerbrey-type analysis was sufficient to accurately interpret the QCM-D results for streptavidin binding, but viscoelastic models were required for vesicle and microsphere binding and for the binding of antibody to the tethered vesicles. Existing viscoelastic models assuming lateral homogeneity could accurately estimate the amount of antibody bound to the tethered vesicles at high surface densities. Osmotic pressure experiments leading to changes in tethered vesicle shape provided significant insight into the effect of interstitial water. In addition, a previously unreported biotin-mediated membrane-membrane interaction was observed.