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.

We report electrochemical impedance spectroscopy measurements to characterize the membrane-disruptive properties of medium-chain fatty acid and monoglyceride mitigants interacting with tethered bilayer lipid membrane (tBLM) platforms composed of E. coli bacterial lipid extracts. The tested mitigants included capric acid (CA) and monocaprin (MC) with 10-carbon long hydrocarbon chains, and lauric acid (LA) and glycerol monolaurate (GML) with 12-carbon long hydrocarbon chains. All four mitigants disrupted E. coli tBLM platforms above their respective critical micelle concentration (CMC) values; however, there were marked differences in the extent of membrane disruption. In general, CA and MC caused larger changes in ionic permeability and structural damage, whereas the membrane-disruptive effects of LA and GML were appreciably smaller. Importantly, the distinct magnitudes of permeability changes agreed well with the known antibacterial activity levels of the different mitigants against E. coli, whereby CA and MC are inhibitory and LA and GML are non-inhibitory. Mechanistic insights obtained from the EIS data help to rationalize why CA and MC are more effective than LA and GML at disrupting E. coli membranes, and these measurement capabilities support the potential of utilizing bacterial lipid-derived tethered lipid bilayers for predictive assessment of antibacterial drug candidates and mitigants.

Membrane-disrupting lactylates are an important class of surfactant molecules that are esterified adducts of fatty acid and lactic acid and possess industrially attractive properties, such as high antimicrobial potency and hydrophilicity. Compared with antimicrobial lipids such as free fatty acids and monoglycerides, the membrane-disruptive properties of lactylates have been scarcely investigated from a biophysical perspective, and addressing this gap is important to build a molecular-level understanding of how lactylates work. Herein, using the quartz crystal microbalance–dissipation (QCM-D) and electrochemical impedance spectroscopy (EIS) techniques, we investigated the real-time, membrane-disruptive interactions between sodium lauroyl lactylate (SLL)—a promising lactylate with a 12-carbon-long, saturated hydrocarbon chain—and supported lipid bilayer (SLB) and tethered bilayer lipid membrane (tBLM) platforms. For comparison, hydrolytic products of SLL that may be generated in biological environments, i.e., lauric acid (LA) and lactic acid (LacA), were also tested individually and as a mixture, along with a structurally related surfactant (sodium dodecyl sulfate, SDS). While SLL, LA, and SDS all had equivalent chain properties and critical micelle concentration (CMC) values, our findings reveal that SLL exhibits distinct membrane-disruptive properties that lie in between the rapid, complete solubilizing activity of SDS and the more modest disruptive properties of LA. Interestingly, the hydrolytic products of SLL, i.e., the LA + LacA mixture, induced a greater degree of transient, reversible membrane morphological changes but ultimately less permanent membrane disruption than SLL. These molecular-level insights support that careful tuning of antimicrobial lipid headgroup properties can modulate the spectrum of membrane-disruptive interactions, offering a pathway to design surfactants with tailored biodegradation profiles and reinforcing that SLL has attractive biophysical merits as a membrane-disrupting antimicrobial drug candidate.

In light of regulatory considerations, there are ongoing efforts to identify Triton X-100 (TX-100) detergent alternatives for use in the biological manufacturing industry to mitigate membrane-enveloped pathogen contamination. Until now, the efficacy of antimicrobial detergent candidates to replace TX-100 has been tested regarding pathogen inhibition in endpoint biological assays or probing lipid membrane disruption in real-time biophysical testing platforms. The latter approach has proven especially useful to test compound potency and mechanism of action, however, existing analytical approaches have been limited to studying indirect effects of lipid membrane disruption such as membrane morphological changes. A direct readout of lipid membrane disruption by TX-100 detergent alternatives would be more practical to obtain biologically relevant information to guide compound discovery and optimization. Herein, we report the use of electrochemical impedance spectroscopy (EIS) to investigate how TX-100 and selected replacement candidates—Simulsol SL 11W (Simulsol) and cetyltrimethyl ammonium bromide (CTAB)—affect the ionic permeability of tethered bilayer lipid membrane (tBLM) platforms. The EIS results revealed that all three detergents exhibited dose-dependent effects mainly above their respective critical micelle concentration (CMC) values while displaying distinct membrane-disruptive behaviors. TX-100 caused irreversible membrane disruption leading to complete solubilization, whereas Simulsol caused reversible membrane disruption and CTAB induced irreversible, partial membrane defect formation. These findings establish that the EIS technique is useful for screening the membrane-disruptive behaviors of TX-100 detergent alternatives with multiplex formatting possibilities, rapid response, and quantitative readouts relevant to antimicrobial functions.

There is broad interest in fabricating cell-membrane-mimicking, hybrid lipid bilayer (HLB) coatings on titanium oxide surfaces for medical implant and drug delivery applications. However, existing fabrication strategies are complex, and there is an outstanding need to develop a streamlined method that can be performed quickly at room temperature. Towards this goal, herein, we characterized the room-temperature deposition kinetics and adlayer properties of one- and two-tail phosphonic acid-functionalized molecules on titanium oxide surfaces in various solvent systems and identified optimal conditions to prepare self-assembled monolayers (SAMs), upon which HLBs could be formed in select cases. Among the molecular candidates, we identified a two-tail molecule that formed a rigidly attached SAM to enable HLB fabrication via vesicle fusion for membrane-based biosensing applications. By contrast, vesicles adsorbed but did not rupture on SAMs composed of one-tail molecules. Our findings support that two-tail phosphonic acid SAMs offer superior capabilities for rapid HLB coating fabrication at room temperature, and these streamlined capabilities could be useful to prepare durable lipid bilayer coatings on titanium-based materials.