An Infrared Nanospectroscopy Technique for the Study of Electric-Field-Induced Molecular Dynamics

Abstract

Static electric fields play a considerable role in a variety of molecular nanosystems as diverse as single-molecule junctions, molecules supporting electrostatic catalysis, and biological cell membranes incorporating proteins. External electric fields can be applied to nanoscale samples with a conductive atomic force microscopy (AFM) probe in contact mode, but typically, no structural information is retrieved. Here we combine photothermal expansion infrared (IR) nanospectroscopy with electrostatic AFM probes to measure nanometric volumes where the IR field enhancement and the static electric field overlap spatially. We leverage the vibrational Stark effect in the polymer poly(methyl methacrylate) for calibrating the local electric field strength. In the relevant case of membrane protein bacteriorhodopsin, we observe electric-field-induced changes of the protein backbone conformation and residue protonation state. The proposed technique also has the potential to measure DC currents and IR spectra simultaneously, insofar enabling the monitoring of the possible interplay between charge transport and other effects.

Keywords: IR nanospectroscopy; electric-field-induced molecular dynamics; electrostatic AFM probe; membrane proteins; vibrational Stark effect.

Figure 1

(a) Schematic of the DC circuit integrated in the AFM-IR setup. (b) Time scheme of the procedure followed for the acquisition of difference-spectroscopy data δA(ν)_i. Δt_on represents the time interval for the AFM-IR spectra acquisition (in Figures 1c and 2h,i Δt_on ∼ 20 s), and Δt_off represents the voltage ramp time plus a settle time for static charge removal (Δt_off ∼ Δt_on). (c) Representative AFM-IR spectra recorded on the 55 nm-thick PMMA film with the flat tip in the absence (black) and in the presence (yellow) of an applied external voltage V. (d) ΔA(ν)_i acquired on the 55 nm-thick PMMA film for different values of the applied voltage V_i (gray dotted curves) and the best VSE fitting curves reported (blue lines). A smoothing spline algorithm has been applied to the AFM-IR data. (e) Schematic representation of the effect of an electric field on the vibrational transition energy in the case of an anharmonic molecular potential (left panel) and of the line shape broadening of an absorption peak for an isotropic sample because of the VSE (right panel). (f) Plot of the second derivative coefficients (empty circles) obtained from the fitting curves in (d) using eq 1, highlighting the linear dependence on .

Figure 2

(a, b) SEM images of the flat tip and the sharp tip, respectively. (c, d) Numerical simulations of the static electric field F_stat applied with the flat tip and the sharp tip, respectively, on the 55 nm-thick PMMA sample for a voltage V = 1.0 V. (e, f) Electromagnetic simulations of the IR field F_IR enhancement at λ = 5.8 μm in the same experimental conditions of (c) and (d). The maps are saturated to the maximum field intensity in the PMMA layer. (g) Representative AFM-IR spectrum acquired on the 23 nm-thick sample with the flat tip and the relative first derivative (continuous black line, 30×) and second derivative (dashed black line, ×150). (h) Comparison of the difference spectra ΔA(ν)_i obtained with the flat tip (gray dotted curves) and (i) with the sharp tip (light-blue dotted curves) on the 55 nm-thick PMMA sample (top panel) and on the 23 nm-thick PMMA sample (bottom panel). A smoothing spline algorithm has been applied to the AFM-IR data.

Figure 3

(a) Sketch reporting μ⃗ and μ⃗_stat for two representative BR molecules belonging to the same trimer. The direction of μ⃗_stat has been obtained using the software described in ref (73) with PDB-ID Code 1FBB and that of μ⃗ from ref (74). (b) Top: AFM topography of the two overlapping cell membranes exposing the CP side on which the AFM-IR measurement has been conducted. Cell membrane patches showing the less rough EC side can also be also identified. Bottom: sketch of two purple membranes placed in the nanogap between the flat tip and ultraflat gold substrate. (c) Amide-I band (green curve) of a representative AFM-IR spectrum acquired on the area pointed out by the black square in (b). (d) ΔA(ν)₀ (black dotted curve) and ΔA(ν)_±3V (gray dotted curves) obtained in the double membrane of panel (b). A smoothing spline algorithm has been applied to the AFM-IR data.

Figure 4

Comparison of two AFM-IR difference spectra (data smoothed with a spline algorithm). The green curve is the light-induced ΔA acquired on a purple membrane stack. The gray curve is an electric-field-induced ΔA(ν)_−3V acquired with a flat AFM tip on a stack of two overlapping purple membranes in high-hydration condition. The blue, yellow, and red shaded regions indicate the spectral range of the features related to retinal photoisomerization, protein conformational changes, and protonation of a carbonyl group, respectively.