Watching individual molecules flex within lipid membranes using SERS

Abstract

Interrogating individual molecules within bio-membranes is key to deepening our understanding of biological processes essential for life. Using Raman spectroscopy to map molecular vibrations is ideal to non-destructively 'fingerprint' biomolecules for dynamic information on their molecular structure, composition and conformation. Such tag-free tracking of molecules within lipid bio-membranes can directly connect structure and function. In this paper, stable co-assembly with gold nano-components in a 'nanoparticle-on-mirror' geometry strongly enhances the local optical field and reduces the volume probed to a few nm(3), enabling repeated measurements for many tens of minutes on the same molecules. The intense gap plasmons are assembled around model bio-membranes providing molecular identification of the diffusing lipids. Our experiments clearly evidence measurement of individual lipids flexing through telltale rapid correlated vibrational shifts and intensity fluctuations in the Raman spectrum. These track molecules that undergo bending and conformational changes within the probe volume, through their interactions with the environment. This technique allows for in situ high-speed single-molecule investigations of the molecules embedded within lipid bio-membranes. It thus offers a new way to investigate the hidden dynamics of cell membranes important to a myriad of life processes.

Figure 1. Formation and characterization of gap plasmon sensor.

(a), Schematic of gold nanoparticle (diameter d = 80 nm) deposited upon lipid-alkanethiol hybrid bilayer on a planar gold surface, in solution. Lipids are POPC:DOTAP and SAM is octadecanethiol. (b), Wide-field scattering image of gold nanoparticles on lipid hybrid layer. (c), Reproducible scattering spectra of the gap plasmon resonance from individual gold particles (shown in inset) on the hybrid lipid layer.

Figure 2. Lipidomics of lipid layers with gap plasmon SERS.

(a), SERS spectra (1 s acquisition time, averaged over 300 subsequent spectra) for three separate particles on a POPC:DOTAP 80:20 hybrid bilayer. (b), SERS spectrum of D-POPC:H-DOTAP (10 s acquisition time) highlighting Raman modes shifted due to deuteration. (c), Selected spectra from repeated measurement of a three component lipid layer including 5% PTPC possessing unique acetylene signature mode highlighted in molecule. Inset: SERS intensity of PTPC acetylene mode with time (normalized to CH₂ stretch intensity and acquisition time 1 s). All spectra in a–c acquired with 1 mW excitation at λ_R = 633 nm.

Figure 3. Fast dynamics of hybrid lipid-bilayer (POPC:DOTAP) SERS.

(a), Time dependent evolution of SERS from a single gold NPoM. Trajectories of individual lines show dramatic jumps and correlated shifts, corresponding to lipid dynamics within the gap plasmon volume. Right: Selected short-time dynamics highlighting the (anti-)/correlations between Raman lines. (b), Raman trajectory, including flexing events labelled I, II extracted to right. (c), Schematic lipid flexing within gap plasmon hot spot. (d), i, Frequency shifts at each time step of Raman line pairs (ν₁, ν₂) showing positive (orange) and negative (purple) correlations. ii, Strength of correlation between pairs of Raman lines (ν₁, ν₂) with size of points giving correlation strength R². iii, Magnitude of correlation (as in ii) when separation between bonds involved increases (dashed linear fit has R²>0.5). All spectra acquired at λ_R = 633 nm with acquisition time of 1 s.