As the threat of antibiotic resistance increases, there is a particular focus on developing antimicrobials against pathogenic bacteria whose multidrug resistance is especially entrenched and concerning. One such target for novel antimicrobials is the ATP-binding cassette (ABC) transporter MsbA that is present in the plasma membrane of Gram-negative pathogenic bacteria where it is fundamental to the survival of these bacteria. Supported lipid bilayers (SLBs) are useful in monitoring membrane protein structure and function since they can be integrated with a variety of optical, biochemical, and electrochemical techniques. Here, we form SLBs containing Escherichia coli MsbA and use atomic force microscopy (AFM) and structured illumination microscopy (SIM) as high-resolution microscopy techniques to study the integrity of the SLBs and incorporated MsbA proteins. We then integrate these SLBs on microelectrode arrays (MEA) based on the conducting polymer poly(3,4-ethylenedioxy-thiophene) poly(styrene sulfonate) (PEDOT:PSS) using electrochemical impedance spectroscopy (EIS) to monitor ion flow through MsbA proteins in response to ATP hydrolysis. These EIS measurements can be correlated with the biochemical detection of MsbA-ATPase activity. To show the potential of this SLB approach, we observe not only the activity of wild-type MsbA but also the activity of two previously characterized mutants along with quinoline-based MsbA inhibitor G907 to show that EIS systems can detect changes in ABC transporter activity. Our work combines a multitude of techniques to thoroughly investigate MsbA in lipid bilayers as well as the effects of potential inhibitors of this protein. We envisage that this platform will facilitate the development of next-generation antimicrobials that inhibit MsbA or other essential membrane transporters in microorganisms.

The use of bacteriophages, viruses that specifically infect bacteria, as antibiotics has become an area of great interest in recent years as the effectiveness of conventional antibiotics recedes. The detection of phage interactions with specific bacteria in a rapid and quantitative way is key for identifying phages of interest for novel antimicrobials. Outer membrane vesicles (OMVs) derived from Gramnegative bacteria can be used to make supported lipid bilayers (SLBs) and therefore in vitro membrane models that contain naturally occurring components of the bacterial outer membrane. In this study, we employed Escherichia coli OMV derived SLBs and use both fluorescent imaging and mechanical sensing techniques to show their interactions with T4 phage. We also integrate these bilayers with microelectrode arrays (MEAs) functionalized with the conducting polymer PEDOT:PSS and show that the pore forming interactions of the phages with the SLBs can be monitored using electrical impedance spectroscopy. To highlight our ability to detect specific phage interactions, we also generate SLBs using OMVs derived from Citrobacter rodentium, which is resistant to T4 phage infection, and identify their lack of interaction with the phage. The work presented here shows how interactions occurring between the phages and these complex SLB systems can be monitored using a range of experimental techniques. We believe this approach can be used to identify phages that work against bacterial strains of interest, as well as more generally to monitor any pore forming structure (such as defensins) interacting with bacterial outer membranes, and thus aid in the development of next generation antimicrobials.

The use of bacteriophage, viruses that specifically infect bacteria, as antibiotics has become an area of great interest in recent years as the effectiveness of conventional antibiotics recedes. The detection of phage interactions with specific bacteria in a rapid and quantitative way is key for identifying phage of interest for novel antimicrobials. Outer membrane vesicles (OMVs) derived from gram-negative bacteria can be used to make supported lipid bilayers (SLBs) and therefore in vitro membrane models that contain naturally occurring components of the bacterial outer membrane. In this study, we used Escherichia coli OMV derived SLBs and use both fluorescent imaging and surface sensitive techniques to show their interactions with T4 phage. We also integrate these bilayers with microelectrode arrays (MEAs) functionalised with the conducting polymer PEDOT:PSS and show that the pore forming interactions of the phage with the SLBs can be monitored using electrical impedance spectroscopy. To highlight our ability to detect specific phage interactions, we also generate SLBs using OMVs derived from Citrobacter rodentium, which is resistant to T4 phage infection, and identify their lack of interaction with phage. The work presented here shows how interactions occurring between phage and these complex SLB systems can be monitored using a range of experimental techniques. We believe this approach can be used to identify phage against bacterial strains of interest, as well as more generally to monitor any pore forming structure (such as defensins) interacting with bacterial outer membranes, and thus aid in the development of next generation antimicrobials.

Tumour-derived extracellular vesicles (TEVs) induce the epithelial-to-mesenchymal (EMT) in non-malignant cells to promote invasion and cancer metastasis. As such, TEVs represent a novel therapeutic target in a field severely lacking in efficacious anti-metastasis treatments. However, scalable technologies that allow continuous, multiparametric monitoring of therapeutic response for identifying metastasis inhibitors are missing. Here, we report the development of a platform based on organic electrochemical transistors (OECTs) for the real-time monitoring of TEV-induced EMT and screening of anti-metastatic drugs. We used TEVs derived from the triple-negative breast cancer (TNBC) cell line MDA-MB-231, to induce EMT in non-malignant breast epithelial cells (MCF10A) over a 9-day period, recapitulating a model of invasive ductal carcinoma metastasis. We performed extensive biological validation using immunofluorescence (IF) imaging and protein expression analysis, providing mechanistic insight using an epigenetics approach, and demonstrate our ability to obtain multiparametric functional readouts of cells using OECTs. Further, by employing OECT-based phenotypic drug screening, we identify heparin as an effective blocker of TEV-induced EMT in vitro, showcasing a promising anti-metastatic drug.

Tumor‐derived extracellular vesicles (TEVs) induce the epithelial‐to‐mesenchymal transition (EMT) in nonmalignant cells to promote invasion and cancer metastasis, representing a novel therapeutic target in a field severely lacking in efficacious antimetastasis treatments. However, scalable technologies that allow continuous, multiparametric monitoring for identifying metastasis inhibitors are absent. Here, the development of a functional phenotypic screening platform based on organic electrochemical transistors (OECTs) for real‐time, noninvasive monitoring of TEV‐induced EMT and screening of antimetastatic drugs is reported. TEVs derived from the triple‐negative breast cancer cell line MDA‐MB‐231 induce EMT in nonmalignant breast epithelial cells (MCF10A) over a nine‐day period, recapitulating a model of invasive ductal carcinoma metastasis. Immunoblot analysis and immunofluorescence imaging confirm the EMT status of TEV‐treated cells, while dual optical and electrical readouts of cell phenotype are obtained using OECTs. Further, heparin, a competitive inhibitor of cell surface receptors, is identified as an effective blocker of TEV‐induced EMT. Together, these results demonstrate the utility of the platform for TEV‐targeted drug discovery, allowing for facile modeling of the transient drug response using electrical measurements, and provide proof of concept that inhibitors of TEV function have potential as antimetastatic drug candidates.