**2. Materials and Methods**

Chemicals. 1,2-dimyristoyl-*sn*-glycero-3-phosphocholine (DMPC) and 3-([3-cholamid opropyl]dimethylammonio)-2-hydroxy-1-propanesulfonate (CHAPSO) were purchased from Avanti Polar Lipids Inc., Alabaster, AL, USA. Sodium fluoride, 1-thio-β-D-glucose, hydrofluoric acid, and sodium tetrachloroaurate were purchased from Sigma-Aldrich Sp. z. o. o., Poznan, Poland. All other reagents and solvents were obtained from Avantor Performance Materials Poland S.A., Gliwice, Poland. All chemicals were used as received. The water was purified through the Milli-Q system (resistivity 18.2 M<sup>Ω</sup> × cm). In all experiments, we have used an aqueous solution of 0.1 M NaF.

Bicelles preparation. Bicelles were prepared according to the protocol described by Ujwal and coworkers [28]. 260 mg of DMPC and 90 mg of CHAPSO were transferred to the vial and mixed with 1 mL of ultrapure Milli-Q water with a resistivity of 18.2 M<sup>Ω</sup> × cm. To disperse lipids and obtain bicelles, the suspension was warmed to 40 ◦C and then cooled to −20 ◦C. The steps with warming and cooling were repeated more than 25 times until the mixture at room temperature was homogeneous and in gel consistency. However, 10 cycles should be enough to observe the formation of the gel at room temperature and cloudiness upon cooling. For the experiments, bicelles were dissolved in 0.1 M NaF solution with a volume ratio of 1:1000.

Electrochemistry. Electrochemical experiments were performed using CHI 650B potentiostat (CH Instruments Inc., Austin, TX, USA) in a three-electrode cell with an Ag|AgCl|sat.KCl reference electrode, a Pt foil counter electrode, and an Au(111) working electrode. The measurements were carried out in a hanging meniscus configuration. Before each experiment, the Au(111) working electrode was cleaned in piranha solution (H2SO4: H2O<sup>2</sup> 3:1 *v*/*v*. CAUTION: piranha reacts violently with organic compounds) for at least 12 h. Then it was thoroughly rinsed with ultrapure water and flame annealed. The monolayers of 1-thio-β-D-glucose (further referenced as thioglucose) were obtained by immersing

the Au(111) electrode in an aqueous solution containing 0.1 mg/mL of the thioglucose for ~2 h. Next, the electrodes were rinsed with ultrapure water and immersed in bicellar suspension for at least 3 h to obtain lipid assembly. For the freeze–thaw treatment, the electrode was taken out from the suspension and covered with a thin film of the supporting electrolyte. Freeze–thaw treatment was carried out by cooling the electrode with deposited lipid film down to −18 ◦C for 2 h and then warming it up to room temperature. A single freeze–thaw cycle was performed for each experiment. AC voltammetry measurements were performed with a scan rate of 5 mV/s. The RMS amplitude of AC perturbation was 10 mV, and a frequency set at 20 Hz. The differential capacitance was derived based on AC voltammograms from the in-phase and out-of-phase components of the AC signal under the assumption that the electrode-electrolyte interface can be treated as a simple RC circuit. Electrochemical impedance spectroscopy (EIS) measurements were carried out within the frequency range of 10−<sup>1</sup> to 10<sup>4</sup> Hz and the amplitude AC perturbation was 10 mV. All measurements were carried at 22 ± 1 ◦C. The potentials reported in this work are referenced to the Ag|AgCl|sat.KCl electrode.

Topography Imaging. Atomic force microscopy (AFM) experiments were performed with Dimension Icon (Bruker Corporation, Billerica, MA, USA). The imaging of the samples was performed using ScanAsyst Fluid probes (Bruker, nominal spring constant 0.7 N/m, tip radius ~20 nm) in PeakForce Tapping mode. The cantilever was periodically modulated with default amplitude at the frequency of 2 kHz. The exact value of the spring constant and the deflection sensitivity for a given probe was carefully calibrated by thermal tuning before each experiment. All images were recorded in an aqueous solution of 0.1 M NaF at the temperature of 22 ± 1 ◦C. In situ imaging was performed on an Au(111) single crystal (MaTecK, GmbH, Julich, Germany) pre-modified with thioglucose monolayer. The quality of the bare Au(111) surface was verified by AFM imaging before each experiment (see Figure S1 in Supplementary Materials). The images were processed using Nanoscope Analysis software version 1.40 (Bruker Corporation, Billerica, MA, USA) and involved flattening with a first-order polynomial function. The protocol of the thioglucose immobilization on gold was the same as for electrochemical measurements. The images were collected immediately after injection of bicellar suspension into the liquid cell of the atomic force microscope. Freeze–thaw treatment was performed by cooling the sample with deposited lipid film down to −18 ◦C and then warming it up to room temperature.

Surface Enhanced Infrared Absorption Spectroscopy. The spectra were recorded with Nicolet iS50 FTIR spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) with MCT-A detector and custom-made single-reflection accessory. The incident angle was 60◦ and the spectral resolution was 4 cm−<sup>1</sup> . The all-glass custom-made spectroelectrochemical cell was used in all experiments with platinum foil serving as a counter electrode and Ag|AgCl|sat.KCl as a reference electrode. The working electrode was a thin gold film deposited on a reflectance plane of a Si hemispherical prism. Deposition of gold was carried out by dropping an aqueous plating solution onto the hydrogen-terminated Si surface. The plating solution was obtained by mixing 100 µL of 0.03 M NaAuCl4·2H2O, 2 mL of 0.15 M Na2SO<sup>3</sup> + 0.05 M Na2S2O3·5H2O + 0.05 M NH4Cl, and 1 mL of 2% HF. After approximately 90 s of plating, the prism was rinsed with ultrapure water to finish the deposition. The deposited film was further modified by dropping an aqueous solution of thioglucose (0.1 mg/mL) onto the gold surface and after 2 h the prism was gently rinsed with ultrapure water. The spectra are displayed in absorbance units defined as A = log(*I*0/*I*), where *I*<sup>0</sup> corresponds to the intensities of IR radiation observed for the reference spectra, while *I* corresponds to the intensity observed for the sample. The reference spectrum was collected for gold film modified with thioglucose. Data processing was performed using Omnic 9 software (Thermo Fisher Scientific, Waltham, MA, USA).
