High-throughput optical quality control of lipid multilayers fabricated by dip-pen nanolithography

Surface supported phospholipid multilayers are promising materials for nanotechnology because of their tendency to self-organize, their innate biocompatibility, the possibility to encapsulate other materials within the multilayers, and the ability to control the multilayer thickness between ∼ 2 and 100 nm during fabrication. Dip-pen nanolithography (DPN) is an atomic force microscopy (AFM) based fabrication method that allows high-throughput fabrication and integration of a variety of micro- and nanostructured materials including lipid multilayers, with areal throughputs on the scale of cm(2) min(-1). Although multilayer thickness is a critical feature that determines the functionality of the lipid multilayer structures (for instance as carriers for other materials as well as optical scattering properties), reliable height characterization by AFM is slow (on the order of µm(2) min(-1)) and a bottleneck in the lithographic process. Here we describe a novel optical method to reliably measure the height of fluorescent multilayers with thicknesses above 10 nm, and widths above the optical diffraction limit based on calibrating the fluorescence intensity using one-time AFM height measurements. This allows large surface areas to be rapidly and quantitatively characterized using a standard fluorescence microscope. Importantly, different pattern dimensions (0D dots, 1D lines or 2D squares) require different calibration parameters, indicating that shape influences the optical properties of the structured lipid multilayers. This method has general implications in the systematic and high-throughput optical characterization of nanostructure-function relationships.