Membrane-protein binding measured with solution-phase plasmonic nanocube sensors

Abstract

We describe a solution-phase sensor of lipid-protein binding based on localized surface plasmon resonance (LSPR) of silver nanocubes. When silica-coated nanocubes are mixed in a suspension of lipid vesicles, supported membranes spontaneously assemble on their surfaces. Using a standard laboratory spectrophotometer, we calibrated the LSPR peak shift due to protein binding to the membrane surface and then characterized the lipid-binding specificity of a pleckstrin homology domain protein.

Figure 1

The physical properties of Ag@SiO₂ core-shell nanocube. (a) & (b) TEM images of Ag@SiO₂ nanocube. (a) is the close-up image of figure (b). (c)~(f) The elemental maps obtained by high-angle annular dark field scanning TEM (HAADF-STEM) with energy dispersive x-ray spectroscopy (EDS). (c) to (f) represent silver, silicon, oxygen, and carbon, respectively. (g) Top: Detection procedure of nanocube sensors. Supported lipid bilayers are formed by vesicle fusion onto the silica surface, and protein binding is monitored by shifts in the LSPR extinction spectrum. Bottom: Typical spectra of membrane coverage and protein binding to the membrane surfaces. Sequential addition of lipid vesicles, BSA, and streptavidin causes LSPR red shifts. (h) Electric field norm (|E|/E₀) in decibel (dB) of a nanocube at resonance (n = 1.33303, λ₀ = 474 nm) computed using finite-element analysis.

Figure 2

Calibration of the nanocube assay. (a) Relation between LSPR shift and number of streptavidin per nanocube (left vertical axis) and surface density (right axis) measured by titration of biotinyl-cap-PE, titration of streptavidin, and fluorescence measurement of streptavidin concentration. Linear fit slopes are reported in Supplementary Table 1. (b) Top: Concentrations of bound and unbound CTB are detected by multi-component FCS. Alexa 594-CTB binds to vesicles (average diameter 120 nm) containing 0.5% G_M1 and 0.5% BODIPY-FL-DHPE lipids. BODIPY-FL-DHPE was used to determine the average number of vesicles diffusing within the excitation spot. Bottom: Binding kinetics measured by multi-component FCS and nanocube assay. (Error bar of FCS, n = 20, mean ± s.d.) CTB surface density was respectively calculated from known vesicle size and LSPR response to protein mass change in streptavidin-biotin systems (0.191 ng mm⁻² nm⁻¹). (c) Binding kinetics of wild-type and R407S K411S mutant of GST-Ste5 PH to different membrane surfaces. Concentrations of GST-Ste5 PH = 1.6 μM; GST-Ste5 PH mutant = 1.6 μM) (d) Equilibrium binding curves of GST-Ste5 PH to bilayers K_d = 0.49 ± 0.33 μM (PI(4,5)P₂ bilayer) and 1.6 ± 0.45 μM (PI(4,5)P₂-free bilayer) (n = 3, mean ± s.e.m.) Error limits of K_d are derived from the statistical error of curve fitting.