Large-scale two-dimensional (2D) arrays of metallic nanostructures formed by thin-film evaporation over hexagonally close-packed polystyrene spheres are established substrates for surface-enhanced Raman spectroscopy (SERS). By using these substrates with an integrated atomic force microscopy (AFM) and inverted Raman spectroscopy system, simultaneous topographical imaging and high-sensitivity chemical mapping can be performed. In this paper, we have used this technique to investigate supported bilayers of long-chain fatty acids and phospholipids deposited by the Langmuir-Blodgett (LB) and spin-coating techniques. Nanosphere lithography (NSL) substrates created from 384 and 1002 nm polystyrene spheres and silver (Ag) deposition on glass and sapphire substrates were characterized for SERS in terms of their structure, distribution, and level of enhancement. SERS mappings of rhodamine 6G (R6G) and p-aminothiophenol (p-ATP) monolayers on the 384 nm substrates demonstrate high and uniform enhancement at a micrometer scale. The enhancement was sufficiently high to enable measurement of SERS spectra for arachidic acid (AA) and dipalmitoylphosphatidylcholine (DPPC) layers on sapphire/Ag substrates. The roughness of these substrates (<2 nm) was lower than for glass/Ag (∼5 nm); therefore, simultaneous to SERS it was possible to measure the topography of the samples by AFM and determine the number of layers of AA and DPPC. This study shows the potential of the combined AFM/SERS technique for spectral and topographical characterization of phospholipid bilayers. This may prove to be an interesting approach for further studies with more complex heterogeneous lipid mixtures aiming to measure spatially resolved features such as microdomains.

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