Mechanistic Evaluation of Antimicrobial Lipid Interactions with Tethered Lipid Bilayers by Electrochemical Impedance Spectroscopy

Abstract

There is extensive interest in developing real-time biosensing strategies to characterize the membrane-disruptive properties of antimicrobial lipids and surfactants. Currently used biosensing strategies mainly focus on tracking membrane morphological changes such as budding and tubule formation, while there is an outstanding need to develop a label-free biosensing strategy to directly evaluate the molecular-level mechanistic details by which antimicrobial lipids and surfactants disrupt lipid membranes. Herein, using electrochemical impedance spectroscopy (EIS), we conducted label-free biosensing measurements to track the real-time interactions between three representative compounds-glycerol monolaurate (GML), lauric acid (LA), and sodium dodecyl sulfate (SDS)-and a tethered bilayer lipid membrane (tBLM) platform. The EIS measurements verified that all three compounds are mainly active above their respective critical micelle concentration (CMC) values, while also revealing that GML induces irreversible membrane damage whereas the membrane-disruptive effects of LA are largely reversible. In addition, SDS micelles caused membrane solubilization, while SDS monomers still caused membrane defect formation, shedding light on how antimicrobial lipids and surfactants can be active in, not only micellar form, but also as monomers in some cases. These findings expand our mechanistic knowledge of how antimicrobial lipids and surfactants disrupt lipid membranes and demonstrate the analytical merits of utilizing the EIS sensing approach to comparatively evaluate membrane-disruptive antimicrobial compounds.

Keywords: antimicrobial lipid; electrochemical impedance spectroscopy; membrane disruption; surfactant; tethered bilayer lipid membrane.

Figure 1

(A) Molecular structures of GML, LA, and SDS. (B) Schematic illustration of EIS measurement setup with the tBLM platform. The corresponding electrical properties of the tethered lipid bilayer are modelled in terms of conductance (G_m) and capacitance (C_m). (C,D) Schematic illustration of (C) membrane thinning/thickening and (D) membrane lysis effects and typical measurement responses.

Figure 2

(A) Time-resolved conductance (G_m) and capacitance (C_m) shifts upon 500 µM GML addition to the tBLM platform. The arrows indicate compound addition at t = 10 min and buffer washing at t = 40 min, respectively. (B) Bode plots for 500 µM GML addition to the tBLM platform, whereby baseline, treatment, and post-washing reflect the initial measurement signal, signal after compound addition, and signal after buffer washing, respectively. Corresponding data for (C,D) 250 µM GML and (E,F) 125 µM GML addition to the tBLM platform.

Figure 3

(A) Time-resolved conductance (G_m) and capacitance (C_m) shifts upon 2000 µM LA addition to the tBLM platform. The arrows indicate compound addition at t = 10 min and buffer washing at t = 40 min, respectively. (B) Bode plots for 2000 µM LA addition to the tBLM platform, whereby baseline, treatment, and post-washing reflect the initial measurement signal, signal after compound addition, and signal after buffer washing, respectively. Corresponding data for (C,D) 1000 µM LA and (E,F) 500 µM LA addition to the tBLM platform.

Figure 4

(A) Time-resolved conductance (G_m) and capacitance (C_m) shifts upon 2000 µM SDS addition to the tBLM platform. The arrows indicate compound addition at t = 10 min and buffer washing at t = 40 min, respectively. (B) Bode plots for 2000 µM SDS addition to the tBLM platform, whereby baseline, treatment, and post-washing reflect the initial measurement signal, signal after compound addition, and signal after buffer washing, respectively. Corresponding data for (C,D) 1000 µM SDS and (E,F) 500 µM SDS addition to the tBLM platform.

Figure 5

(A) Time-resolved conductance (G_m) and capacitance (C_m) shifts upon 250 µM SDS addition to the tBLM platform. The arrows indicate compound addition at t = 10 min and buffer washing at t = 40 min, respectively. (B) Bode plots for 250 µM SDS addition to the tBLM platform, whereby baseline, treatment, and post-washing reflect the initial measurement signal, signal after compound addition, and signal after buffer washing, respectively. Corresponding data for (C,D) 125 µM SDS and (E,F) 63 µM SDS addition to the tBLM platform.

Figure 6

Schematic summary illustrating the membrane-disruptive effects of (A) SDS, (B) GML, and (C) LA micelles on tethered bilayer lipid membrane platforms. The inferred scenarios reflect the EIS measurement trends observed in each case and are proposed based on the wealth of antimicrobial peptide data that has been obtained for tBLM platform experiments using the EIS technique in past reports [33,36].