Breaking sensitivity limits of QCM-D: An integrated nano-honeycomb sensor for characterization molecular interactions with lipid bilayers.

Abstract

In the last few years Quartz Crystal Microbalance with Dissipation (QCM-D) emerged as an indispensable label-free method for monitoring formation of lipid bilayers on surfaces and assessing quality of the bilayers formed. QCM-D provides time-resolved data on mass and viscoelastic properties of molecular layers at the sensor surface and therefore is suitable for detecting vesicle adhesion, rupture, and spreading to form planar surface-supported bilayers. However, the small surface areas of QCM sensors impose severe limitation on mass sensitivity of the method. This is, perhaps, the main reason why QCM-D is rarely employed for studying lipid bilayer phase transitions and membrane binding events. To overcome these limitations, we have integrated the QCM sensor with an array of rigid honeycomb-packed anodic aluminum oxide (AAO) nanopores of ca. 25-80 nm in diameter and 2-10 μm in length by anodizing aluminum film deposited on the electrode surface. The resulting nanostructured QCM sensor increased surface area and improved sensitivity of the method by >100-fold. AAO-QCM electrode also provided a nanoscale template for self-assembly of lipid bilayers into nanotubular structures without any chemical attachment. We then employed such nanopore-confined bilayers to observe the main and more difficult to characterize pre-transition of membranes prepared from either pure 1,2-dimyristoyl-sn-glycerol-3-phosphocholine (DMPC) or 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC) lipids. Observed changes in QCM frequency reflected variations in lipid density upon phase transitions and occurred at temperatures in agreement with literature data. The signal-to-noise ratio of the phase transition peaks increased by 300-fold when compared to conventional QCM-D sensors. To further explore potential applications of AAO-QCM-D, ethanol-induced biphasic effects of phospholipid bilayers were studied. High sensitivity of AAO-QCM electrodes makes the method highly attractive for developing biosensing applications based on detecting changes in lipid packing at the nanoscale.