Vesicle fusion studied by surface plasmon resonance and surface plasmon fluorescence spectroscopy

Abstract

Substrate-supported planar lipid bilayers are generated most commonly by the adsorption and transformation of phospholipid vesicles (vesicle fusion). We have recently demonstrated that simultaneous measurements of surface plasmon resonance (SPR) and surface plasmon fluorescence spectroscopy (SPFS) are highly informative for monitoring lipid membranes on solid substrates. SPR and SPFS provide information on the amount and topography of adsorbed lipid membranes, respectively. In this study, the vesicle fusion process was studied in detail by measuring SPR-SPFS at a higher rate and plotting the obtained fluorescence intensity versus film thickness. We could track the initial adsorption of vesicles, the onset of vesicle rupture occurring at certain vesicle coverage of the surface, and the autocatalytic transformation into planar bilayers. We also monitored vesicle fusion of the same vesicle suspensions by quartz crystal microbalance with dissipation monitoring (QCM-D). We compared the results obtained from SPR-SPFS and QCM-D to highlight the unique information provided by SPR-SPFS.

FIGURE 1

Schematic illustration of vesicle fusion on silica substrates. Left: Adsorbed vesicles transform into a planar bilayer. Right: Silica-coated substrate used in this study.

FIGURE 2

Schematic diagram of the SPR-SPFS setup.

FIGURE 3

SPR (lower frame) and SPFS (upper frame) spectra measured in buffer solution before (dashed lines) and after (solid lines) the incubation with the vesicle suspension of POPC/POPS (4:1). The total lipid concentration of vesicle suspensions was 0.1 mM.

FIGURE 4

Kinetic observation of vesicle fusion by SPR-SPFS. The lipid layer thickness observed by SPR (lower frame) and the fluorescence intensity observed by SPFS (upper frame) were plotted versus incubation time for vesicles of (a) POPC, (b) POPC/POPS (4:1), and (c) POPC/DOTAP (4:1). A trace amount of DiD (10⁻⁴ mol/mol) was added to all samples. The total concentration of vesicle suspensions was 0.1 mM. The solid columns indicate values after successive rinsings with the buffer solution, Milli-Q water, and the buffer solution, respectively (from left to right). Milli-Q water and buffer solution have different refractive indices. This difference was incorporated in the SPR curve fitting to evaluate the adsorbed layer thickness.

FIGURE 5

Plot of fluorescence intensity versus adsorbed lipid film thickness measured by SPR-SPFS during the vesicle fusion process. Four types of vesicles were compared: POPC (open squares); POPC/POPS (4:1) (solid circles); POPC/POPS (1:1) (solid squares); POPC/DOTAP (4:1) (solid triangles). The arrows indicate (a) before the addition of vesicles, (b) initial linear phase (adsorption of vesicles), (c) onset of vesicle rupture (for POPC/POPS (4:1)), (d) complete coverage with planar bilayer, and (e) additional adsorption of vesicles (for POPC), respectively. The sign (f) indicates the profile of POPC/POPS (1:1). The dotted and dashed lines were given to indicate the linearity of fluorescence intensity versus film thickness for adsorbed vesicles and planar bilayers, respectively.

FIGURE 6

The amount of adsorbed vesicle and planar bilayer during the vesicle fusion of POPC/POPS (4:1) estimated from the plot in Fig. 5. The open squares are the total film thickness determined by SPR. The solid circles and triangles are the estimated film thickness for vesicles and planar bilayers, respectively. (The film thickness determined by SPR is slightly underestimated for vesicles.)

FIGURE 7

Kinetic measurements by SPR-SPFS and QCM-D for the vesicle fusion of POPC/POPS (4:1): The dissipation (ΔD) and frequency shift (Δf) measured by QCM-D were plotted versus vesicle incubation time, together with the fluorescence intensity (SPFS: upper frame) and the lipid bilayer thickness (SPR: lower frame), respectively. The estimated amounts of adsorbed vesicle and planar bilayer (same as in Fig. 6) are also given for comparison with the QCM-D data. The arrows with a sign “C” indicate the critical point at which rupture of vesicles started (determined from the plot in Fig. 5).