An Impedance-Based Approach for Sensing Cyclodextrin-Mediated Modulation of Membrane Cholesterol.

Cyclodextrin molecules are increasingly being used in biological research and as therapeutic agents to alter membrane cholesterol content, yet there is much to learn about their interactions with cell membranes. We present a biomembrane-based organic electronic platform capable of detecting interactions of cell membrane constituents with methyl-ß-cyclodextrin (MßCD). This approach enables label-free sensing and quantification of changes in membrane integrity resulting from such interactions. In this work, we employ cholesterol-containing supported lipid bilayers (SLBs) formed on conducting polymer-coated electrodes to investigate how MßCD impacts membrane resistance. By examining the outcomes of MßCD interactions with SLBs of varying cholesterol content, we demonstrate that changes in membrane permeability or resistance can be used as a functional measure for predicting cyclodextrin-mediated cholesterol extraction from cellular membranes. Furthermore, we use the SLB platforms to electronically monitor cholesterol delivery to membranes following exposure to MßCD pre-loaded with cholesterol, observing that cholesterol enrichment is commensurate with an increase in resistance. This biomembrane-based bioelectronic sensing system offers a tool to quantify the modulation of membrane cholesterol content using membrane resistance and provides information regarding MßCD-mediated changes in membrane integrity. Given the importance of membrane integrity for barrier function in cells, such knowledge is essential for our fundamental understanding of MßCD as a membrane cholesterol modulator and therapeutic delivery vehicle.

Gram-Positive Bacterial Membrane-Based Biosensor for Multimodal Investigation of Membrane-Antibiotic Interactions.

As membrane-mediated antibiotic resistance continues to evolve in Gram-positive bacteria, the development of new approaches to elucidate the membrane properties involved in antibiotic resistance has become critical. Membrane vesicles (MVs) secreted by the cytoplasmic membrane of Gram-positive bacteria contain native components, preserving lipid and protein diversity, nucleic acids, and sometimes virulence factors. Thus, MV-derived membrane platforms present a great model for Gram-positive bacterial membranes. In this work, we report the development of a planar bacterial cytoplasmic membrane-based biosensor using MVs isolated from the Bacillus subtilis WT strain that can be coated on multiple surface types such as glass, quartz crystals, and polymeric electrodes, fostering the multimodal assessment of drug-membrane interactions. Retention of native membrane components such as lipoteichoic acids, lipids, and proteins is verified. This biosensor replicates known interaction patterns of the antimicrobial compound, daptomycin, with the Gram-positive bacterial membrane, establishing the applicability of this platform for carrying out biophysical characterization of the interactions of membrane-acting antibiotic compounds with the bacterial cytoplasmic membrane. We report changes in membrane viscoelasticity and permeability that correspond to partial membrane disruption when calcium ions are present with daptomycin but not when these ions are chelated. This biomembrane biosensing platform enables an assessment of membrane biophysical characteristics during exposure to antibiotic drug candidates to aid in identifying compounds that target membrane disruption as a mechanism of action.

Bioelectronic Platform to Investigate Charge Transfer between Photoexcited Quantum Dots and Microbial Outer Membranes.

Photosynthetic semiconductor biohybrids (PSBs) convert light energy to chemical energy through photo-driven charge transfer from nanocrystals to microorganisms that perform bioreactions of interest. Initial proof-of-concept PSB studies with an emphasis on enhanced CO2 conversion have been encouraging; however, bringing the broad prospects of PSBs to fruition is contingent on establishing a firm fundamental understanding of underlying interfacial charge transfer processes. We introduce a bioelectronic platform that reduces the complexity of PSBs by focusing explicitly on interactions between colloidal quantum dots (QDs), microbial outer membranes, and native, small-molecule redox mediators. Our model platform employs a standard three-electrode electrochemical cell with supported outer membranes of Pseudomonas aeruginosa, pyocyanin redox mediators, and semiconducting CdSe QDs dispersed in an aqueous electrolyte. We present a comprehensive electrochemical analysis of this platform via electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV), and chronoamperometry (CA). EIS reveals the formation and electronic properties of supported outer membrane films. CV reveals the electrochemically active surface area of P. aeruginosa outer membranes and that pyocyanin is the sole species that performs redox with these outer membranes under sweeping applied potential. CA demonstrates that photoexcited charge transfer in this system is driven by the reduction of pyocyanin at the QD surface followed by diffusion of reduced pyocyanin through the outer membrane. The broad applicability of this platform across many bacterial species, QD architectures, and controlled environmental conditions affords the possibility to define design principles for future PSB systems to synergistically integrate concurrent advances in genetically engineered organisms and inorganic nanomaterials.

Impedance sensing of antibiotic interactions with a pathogenic E. coli outer membrane supported bilayer.

Antibiotic resistance is a growing global health concern due to the decreasing number of antibiotics available for therapeutic use as more drug-resistant bacteria develop. Changes in the membrane properties of Gram-negative bacteria can influence their response to antibiotics and give rise to resistance. Thus, understanding the interactions between the bacterial membrane and antibiotics is important for elucidating microbial membrane properties to use for designing novel antimicrobial drugs. To study bacterial membrane-antibiotic interactions, we created a surface-supported planar bacterial outer membrane model on an optically-transparent, conducting polymer surface (poly (3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS)). This model enables membrane characterization using fluorescence microscopy and electrochemical impedance spectroscopy (EIS). The membrane platform is fabricated using outer membrane vesicles (OMVs) isolated from clinically relevant Gram-negative bacteria, enterohemorrhagic Escherichia coli. This approach enables us to mimic the native components of the bacterial membrane by incorporating native lipids, membrane proteins, and lipopolysaccharides. Using EIS, we determined membrane impedance and captured membrane-antibiotic interactions using the antibiotics polymyxin B, bacitracin, and meropenem. This sensor platform incorporates aspects of the biological complexity found in bacterial outer membranes and, by doing so, offers a powerful, biomimetic approach to the study of antimicrobial drug interactions.

Detection of Ganglioside-Specific Toxin Binding with Biomembrane-Based Bioelectronic Sensors.

Gangliosides, glycolipids that are abundant in the plasma membrane outer leaflet, play an integral role in cellular recognition, adhesion, and infection by interacting with different endogenous molecules, viruses, and toxins. Model membrane systems, such as ganglioside-enriched supported lipid bilayers (SLBs), present a useful tool for sensing, characterizing, and quantifying such interactions. In this work, we report the formation of ganglioside GM1-rich SLBs on conducting polymer electrodes using a solvent-assisted lipid bilayer assembly method to investigate changes in membrane electrical properties upon binding of the B subunit of cholera toxin. The sensing capabilities of our platform were investigated by varying both the receptor and the toxin concentrations in the system as well as using a complex sample (milk contaminated with the toxin) and monitoring the changes in the electrical properties of the membrane. Our work highlights the potential of such conducting polymer-supported biomembrane-based platforms for detecting the toxins within a complex environment, studying ganglioside-specific biomolecular interactions with toxins and screening inhibitory molecules to prevent these interactions.