Structural Analysis of Lipid Membrane Probes by Enhanced Raman Scattering
Hughes, Hannah Jacqueline
Doctor of Philosophy
Fluorescent membrane probes are one of the many tools used to study the complex structure and processes of cell membranes. The detailed molecular positions and orientations of membrane fluorophores are not well known. These parameters are of interest because they could influence how the fluorescent signal is interpreted. Furthermore, some membrane probes behave differently in different membrane systems, motivating the need to understand how the lipid environment affects the probes orientation and signal. Surface enhanced Raman spectroscopy was used to study the molecular positions and orientations of fluorescent membrane probes embedded in a lipid bilayer in a natural fluid environment. Gold nanostructures can act as an antenna, focusing and enhancing electromagnetic radiation at their surface. When gold nanorods are excited by electromagnetic radiation at their plasmon resonant frequency, the nanoparticle’s free electron gas oscillates, leading to surface enhanced Raman spectroscopy (SERS). SERS a distance dependent enhancement of the Raman scattering signal the allows the possibility to visualize molecular structure. This project utilizes gold nanorods, tuned to an excitation laser wavelength of 785 nm, as a substrate for lipid membranes so that their structure can be analyzed by SERS. As synthesized, gold nanorods are stabilized by the surfactant CTAB. The rods are resuspended in a solution containing a 9:1 molar ratio of DOPC and DOPG, which replace the CTAB to form a well ordered, fluid bilayer electrostatically bound to the gold nanorod surface. Once a lipid bilayer was exchanged onto the gold nanorod surface, a lipid solution interspersed with fluorescent membrane probes was exchanged onto the nanorod surface. The presence of fluorescent probes in the bilayer was confirmed by identifying peak corresponding to the probes in the SERS spectra. The structural analysis by enhanced Raman scattering (SABERS) method combines SERS and unenhanced Raman spectra with theoretical calculations of the optical field and molecular polarizability. Raman measurements of the samples are orientationally averaged, while SERS spectra contain information on molecular position and orientation relative to the surface. Together these reveal the molecular orientation and position of fluorescent membrane probes in phospholipid bilayers. This method offers an approach to analyzing lipid membrane molecular structure under ambient conditions, with microscopic quantities, and without molecular labels.
surface enhanced Raman spectroscopy (SERS), plasmonics