Organic Bioelectronics for In Vitro Systems.

Bioelectronics have made strides in improving clinical diagnostics and precision medicine. The potential of bioelectronics for bidirectional interfacing with biology through continuous, label-free monitoring on one side and precise control of biological activity on the other has extended their application scope to in vitro systems. The advent of microfluidics and the considerable advances in reliability and complexity of in vitro models promise to eventually significantly reduce or replace animal studies, currently the gold standard in drug discovery and toxicology testing. Bioelectronics are anticipated to play a major role in this transition offering a much needed technology to push forward the drug discovery paradigm. Organic electronic materials, notably conjugated polymers, having demonstrated technological maturity in fields such as solar cells and light emitting diodes given their outstanding characteristics and versatility in processing, are the obvious route forward for bioelectronics due to their biomimetic nature, among other merits. This review highlights the advances in conjugated polymers for interfacing with biological tissue in vitro, aiming ultimately to develop next generation in vitro systems. We showcase in vitro interfacing across multiple length scales, involving biological models of varying complexity, from cell components to complex 3D cell cultures. The state of the art, the possibilities, and the challenges of conjugated polymers toward clinical translation of in vitro systems are also discussed throughout.

Transposition into replicating DNA occurs through interaction with the processivity factor.

The bacterial transposon Tn7 directs transposition into actively replicating DNA by a mechanism involving the transposon-encoded protein TnsE. Here we show that TnsE physically and functionally interacts with the processivity factor of the DNA replication machinery in vivo and in vitro. Our work establishes an in vitro TnsABC+E transposition reaction reconstituted from purified proteins and target DNA structures. Using the in vitro reaction we confirm that the processivity factor specifically reorders TnsE-mediated transposition events on target DNAs in a way that matches the bias with active DNA replication in vivo. The TnsE interaction with an essential and conserved component of the replication machinery, and a DNA structure reveals a mechanism by which Tn7, and probably other elements, selects target sites associated with DNA replication.

An attacin antimicrobial peptide, Hill_BB_C10074, from Hermetia illucens with anti-Pseudomonas aeruginosa activity.

BACKGROUND: There is a global need to develop new therapies to treat infectious diseases and tackle the rise in antimicrobial resistance. To date, the larvae of the Black Solider Fly, Hermetia illucens, have the largest repertoire of antimicrobial peptides derived from insects. Antimicrobial peptides are of particular interest in the exploration of alternative antimicrobials due to their potent action and reduced propensity to induce resistance compared with more traditional antibiotics. RESULTS: The predicted attacin from H. illucens, Hill_BB_C10074, was first identified in the transcriptome of H. illucens populations that had been fed a plant-oil based diet. In this study, recombinant Hill_BB_C10074 (500 µg/mL), was found to possess potent antimicrobial activity against the serious Gram-negative pathogen, Pseudomonas aeruginosa. Sequence and structural homology modelling predicted that Hill_BB_C10074 formed a homotrimeric complex that may form pores in the Gram-negative bacterial outer membrane. In vitro experiments defined the antimicrobial action of Hill_BB_C10074 against P. aeruginosa and transmission electron microscopy and electrochemical impedance spectroscopy confirmed the outer membrane disruptive power of Hill_BB_C10074 which was greater than the clinically relevant antibiotic, polymyxin B. CONCLUSIONS: Combining predictive tools with in vitro approaches, we have characterised Hill_BB_C10074 as an important insect antimicrobial peptide and promising candidate for the future development of clinical antimicrobials.

Nanoscale Features of Tunable Bacterial Outer Membrane Models Revealed by Correlative Microscopy.

The rise of antibiotic resistance is a growing worldwide human health issue, with major socioeconomic implications. An understanding of the interactions occurring at the bacterial membrane is crucial for the generation of new antibiotics. Supported lipid bilayers (SLBs) made from reconstituted lipid vesicles have been used to mimic these membranes, but their utility has been restricted by the simplistic nature of these systems. A breakthrough in the field has come with the use of outer membrane vesicles derived from Gram-negative bacteria to form SLBs, thus providing a more physiologically relevant system. These complex bilayer systems hold promise but have not yet been fully characterized in terms of their composition, ratio of natural to synthetic components, and membrane protein content. Here, we use correlative atomic force microscopy (AFM) with structured illumination microscopy (SIM) for the accurate mapping of complex lipid bilayers that consist of a synthetic fraction and a fraction of lipids derived from Escherichia coli outer membrane vesicles (OMVs). We exploit the high resolution and molecular specificity that SIM can offer to identify areas of interest in these bilayers and the enhanced resolution that AFM provides to create detailed topography maps of the bilayers. We are thus able to understand the way in which the two different lipid fractions (natural and synthetic) mix within the bilayers, and we can quantify the amount of bacterial membrane incorporated into the bilayer. We prove the system's tunability by generating bilayers made using OMVs engineered to contain a green fluorescent protein (GFP) binding nanobody fused with the porin OmpA. We are able to directly visualize protein-protein interactions between GFP and the nanobody complex. Our work sets the foundation for accurately understanding the composition and properties of OMV-derived SLBs to generate a high-resolution platform for investigating bacterial membrane interactions for the development of next-generation antibiotics.

Advances in microfluidic in vitro systems for neurological disease modeling.

Neurological disorders are the leading cause of disability and the second largest cause of death worldwide. Despite significant research efforts, neurology remains one of the most failure-prone areas of drug development. The complexity of the human brain, boundaries to examining the brain directly in vivo, and the significant evolutionary gap between animal models and humans, all serve to hamper translational success. Recent advances in microfluidic in vitro models have provided new opportunities to study human cells with enhanced physiological relevance. The ability to precisely micro-engineer cell-scale architecture, tailoring form and function, has allowed for detailed dissection of cell biology using microphysiological systems (MPS) of varying complexities from single cell systems to "Organ-on-chip" models. Simplified neuronal networks have allowed for unique insights into neuronal transport and neurogenesis, while more complex 3D heterotypic cellular models such as neurovascular unit mimetics and "Organ-on-chip" systems have enabled new understanding of metabolic coupling and blood-brain barrier transport. These systems are now being developed beyond MPS toward disease specific micro-pathophysiological systems, moving from "Organ-on-chip" to "Disease-on-chip." This review gives an outline of current state of the art in microfluidic technologies for neurological disease research, discussing the challenges and limitations while highlighting the benefits and potential of integrating technologies. We provide examples of where such toolsets have enabled novel insights and how these technologies may empower future investigation into neurological diseases.

Advances in modelling the human microbiome-gut-brain axis in vitro.

The human gut microbiome has emerged as a key player in the bidirectional communication of the gut-brain axis, affecting various aspects of homeostasis and pathophysiology. Until recently, the majority of studies that seek to explore the mechanisms underlying the microbiome-gut-brain axis cross-talk, relied almost exclusively on animal models, and particularly gnotobiotic mice. Despite the great progress made with these models, various limitations, including ethical considerations and interspecies differences that limit the translatability of data to human systems, pushed researchers to seek for alternatives. Over the past decades, the field of in vitro modelling of tissues has experienced tremendous growth, thanks to advances in 3D cell biology, materials, science and bioengineering, pushing further the borders of our ability to more faithfully emulate the in vivo situation. The discovery of stem cells has offered a new source of cells, while their use in generating gastrointestinal and brain organoids, among other tissues, has enabled the development of novel 3D tissues that better mimic the native tissue structure and function, compared with traditional assays. In parallel, organs-on-chips technology and bioengineered tissues have emerged as highly promising alternatives to animal models for a wide range of applications. Here, we discuss how recent advances and trends in this area can be applied in host-microbe and host-pathogen interaction studies. In addition, we highlight paradigm shifts in engineering more robust human microbiome-gut-brain axis models and their potential to expand our understanding of this complex system and hence explore novel, microbiome-based therapeutic approaches.

Materials for blood brain barrier modeling in vitro.

Brain homeostasis relies on the selective permeability property of the blood brain barrier (BBB). The BBB is formed by a continuous endothelium that regulates exchange between the blood stream and the brain. This physiological barrier also creates a challenge for the treatment of neurological diseases as it prevents most blood circulating drugs from entering into the brain. In vitro cell models aim to reproduce BBB functionality and predict the passage of active compounds through the barrier. In such systems, brain microvascular endothelial cells (BMECs) are cultured in contact with various biomaterial substrates. However, BMEC interactions with these biomaterials and their impact on BBB functions are poorly described in the literature. Here we review the most common materials used to culture BMECs and discuss their potential impact on BBB integrity in vitro. We investigate the biophysical properties of these biomaterials including stiffness, porosity and material degradability. We highlight a range of synthetic and natural materials and present three categories of cell culture dimensions: cell monolayers covering non-degradable materials (2D), cell monolayers covering degradable materials (2.5D) and vascularized systems developing into degradable materials (3D).

Comparison of different anti-Ki67 antibody clones and hot-spot sizes for assessing proliferative index and grading in pancreatic neuroendocrine tumours using manual and image analysis.

AIMS: Ki67 proliferative index (PI) is essential for grading gastroenteric and pancreatic neuroendocrine tumours (GEP NETs). Analytical and preanalytical variables can affect Ki67 PI. In contrast to counting methodology, until now little attention has focused on the question of clone equivalence and the effect of hot-spot size on Ki67 PI in GEP NETs. Using manual counting and image analysis, this study compared the Ki67 PI achieved using MM1, K2 and 30-9 to MIB1, a clone which has been validated for, and is referenced in, guidelines relating to assessment of Ki67 PI in GEP NETs. METHODS AND RESULTS: Forty-two pancreatic NETs were each immunohistochemically stained for the anti-Ki67 clones MIB1, MM1, K2 and 30-9. Ki67 PI was calculated manually and by image analysis, the latter using three different hot-spot sizes. In manual comparisons using single hot-spot high-power fields, non-MIB1 clones overestimated Ki67 PI compared to MIB1, resulting in grading discordances. Image analysis shows good agreement with manual Ki67 PI but a tendency to overestimate absolute Ki67 PI. Increasing the size of tumour hot-spot from 500 to 2000 cells resulted in a decrease in Ki67 PI. CONCLUSION: Different anti-Ki67 clones do not produce equivalent PIs in GEP NETs, and clone selection may therefore affect patient care. Increasing the hot-spot size decreases the Ki67 PI. Greater standardisation in terms of antibody clone selection and hot-spot size is required for grading GEP NETs. Image analysis is an effective tool for assisting Ki67 assessment and allows easier standardisation of the size of the tumour hot-spot.

A highly sensitive molecular structural probe applied to in situ biosensing of metabolites using PEDOT:PSS.

A large amount of research within organic biosensors is dominated by organic electrochemical transistors (OECTs) that use conducting polymers such as poly(3,4-ethylene dioxythiophene) doped with poly(styrenesulfonate) (PEDOT:PSS). Despite the recent advances in OECT-based biosensors, the sensing is solely reliant on the amperometric detection of the bioanalytes. This is typically accompanied by large undesirable parasitic electrical signals from the electroactive components in the electrolyte. Herein, we present the use of in situ resonance Raman spectroscopy to probe subtle molecular structural changes of PEDOT:PSS associated with its doping level. We demonstrate how such doping level changes of PEDOT:PSS can be used, for the first time, on operational OECTs for sensitive and selective metabolite sensing while simultaneously performing amperometric detection of the analyte. We test the sensitivity by molecularly sensing a lowest glucose concentration of 0.02 mM in phosphate-buffered saline solution. By changing the electrolyte to cell culture media, the selectivity of in situ resonance Raman spectroscopy is emphasized as it remains unaffected by other electroactive components in the electrolyte. The application of this molecular structural probe highlights the importance of developing biosensing probes that benefit from high sensitivity of the material's structural and electrical properties while being complimentary with the electronic methods of detection.

Self-Assembly of Mammalian-Cell Membranes on Bioelectronic Devices with Functional Transmembrane Proteins.

Transmembrane proteins (TMPs) regulate processes occurring at the cell surface and are essential gatekeepers of information flow across the membrane. TMPs are difficult to study, given the complex environment of the membrane and its influence on protein conformation, mobility, biomolecule interaction, and activity. For the first time, we create mammalian biomembranes supported on a transparent, electrically conducting polymer surface, which enables dual electrical and optical monitoring of TMP function in its native membrane environment. Mammalian plasma membrane vesicles containing ATP-gated P2X2 ion channels self-assemble on a biocompatible polymer cushion that transduces the changes in ion flux during ATP exposure. This platform maintains the complexity of the native plasma membrane, the fluidity of its constituents, and protein orientation critical to ion channel function. We demonstrate the dual-modality readout using microscopy to characterize protein mobility by single-particle tracking and sensing of ATP gating of P2X2 using electrical impedance spectroscopy. This measurement of TMP activity important for pain sensing, neurological activity, and sensory activity raises new possibilities for drug screening and biosensing applications.

Nanostructured conducting polymers for stiffness controlled cell adhesion.

We propose a facile and reproducible method, based on ultra thin porous alumina membranes, to produce cm(2) ordered arrays of nano-pores and nano-pillars on any kind of substrates. In particular our method enables the fabrication of conducting polymers nano-structures, such as poly[3,4-ethylenedioxythiophene]:poly[styrene sulfonate] ( PEDOT: PSS). Here, we demonstrate the potential interest of those templates with controlled cell adhesion studies. The triggering of the eventual fate of the cell (proliferation, death, differentiation or migration) is mediated through chemical cues from the adsorbed proteins and physical cues such as surface energy, stiffness and topography. Interestingly, as well as through material properties, stiffness modifications can be induced by nano-topography, the ability of nano-pillars to bend defining an effective stiffness. By controlling the diameter, length, depth and material of the nano-structures, one can possibly tune the effective stiffness of a (nano) structured substrate. First results indicate a possible change in the fate of living cells on such nano-patterned devices, whether they are made of conducting polymer (soft material) or silicon (hard material).

Optimization of a planar all-polymer transistor for characterization of barrier tissue.

The organic electrochemical transistor (OECT) is a unique device that shows great promise for sensing in biomedical applications such as monitoring of the integrity of epithelial tissue. It is a label-free sensor that is amenable to low-cost production by roll-to-roll or other printing technologies. Herein, the optimization of a planar OECT for the characterization of barrier tissue is presented. Evaluation of surface coating, gate biocompatibility and performance, and optimization of the geometry of the transistor are highlighted. The conducting polymer poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate), which is used as the active material in the transistor, has the added advantage of allowing significant light transmission compared to traditional electrode materials and thus permits high-quality optical microscopy. The combination of optical and electronic monitoring of cells shown herein provides the opportunity to couple two very complementary techniques to yield a low-cost method for in vitro cell sensing.

Validation of the organic electrochemical transistor for in vitro toxicology.

BACKGROUND: The gastrointestinal epithelium provides a physical and biochemical barrier to the passage of ions and small molecules; however this barrier may be breached by pathogens and toxins. The effect of individual pathogens/toxins on the intestinal epithelium has been well characterized: they disrupt barrier tissue in a variety of ways, such as by targeting tight junction proteins, or other elements of the junctions between adjacent cells. A variety of methods have been used to characterize disruption in barrier tissue, such as immunofluorescence, permeability assays and electrical measurements of epithelia resistance, but these methods remain time consuming, costly and ill-suited to diagnostics or high throughput toxicology. METHODS: The advent of organic electronics has created a unique opportunity to interface the worlds of electronics and biology, using devices such as the organic electrochemical transistor (OECT), whose low cost materials and potential for easy fabrication in high throughput formats represent a novel solution for assessing epithelial tissue integrity. RESULTS: In this study, OECTs were integrated with gastro-intestinal cell monolayers to study the integrity of the gastrointestinal epithelium, providing a very sensitive way to detect minute changes in ion flow across the cell layer due to inherent amplification by the transistor. MAJOR CONCLUSIONS: We validate the OECT against traditional methods by monitoring the effect of toxic compounds on epithelial tissue. We show a systematic characterization of this novel method, alongside existing methods used to assess barrier tissue function. GENERAL SIGNIFICANCE: The toxic compounds induce a dramatic disruption of barrier tissue, and the OECT measures this disruption with increased temporal resolution and greater or equal sensitivity when compared with existing methods. This article is part of a Special Issue entitled Organic Bioelectronics - Novel Applications in Biomedicine.

Probing the specific ion effects of biocompatible hydrated choline ionic liquids on lactate oxidase biofunctionality in sensor applications.

This paper presents an extended study on the ion effects of a series of biocompatible hydrated choline based ionic liquids (ILs) on lactate oxidase (LOx), an important enzyme in biosensing technology for the in vitro detection of lactic acid. Secondary structural analysis revealed changes in the protein conformation in hydrated ILs, while thermal unfolding/aggregation dynamics showed different profiles in the presence or absence of ILs. Moreover, LOx thermally denaturised at 90 °C showed residual activity in the presence of chloride and dihydrogen phosphate anions. Kinetic and lifetime studies were also performed, providing a better understanding of the ion effects of ILs on the biocatalytic activity of the enzyme.

Lab-on-chip technologies for exploring the gut-immune axis in metabolic disease.

The continued rise in metabolic diseases such as obesity and type 2 diabetes mellitus poses a global health burden, necessitating further research into factors implicated in the onset and progression of these diseases. Recently, the gut-immune axis, with diet as a main regulator, has been identified as a possible role player in their development. Translation of conventional 2D in vitro and animal models is however limited, while human studies are expensive and preclude individual mechanisms from being investigated. Lab-on-chip technology therefore offers an attractive new avenue to study gut-immune interactions. This review provides an overview of the influence of diet on gut-immune interactions in metabolic diseases and a critical analysis of the current state of lab-on-chip technology to study this axis. While there has been progress in the development of "immuno-competent" intestinal lab-on-chip models, with studies showing the ability of the technology to provide mechanical cues, support longer-term co-culture of microbiota and maintain in vivo-like oxygen gradients, platforms which combine all three and include intestinal and immune cells are still lacking. Further, immune cell types and inclusion of microenvironment conditions which enable in vivo-like immune cell dynamics as well as host-microbiome interactions are limited. Future model development should focus on combining these conditions to create an environment capable of hosting more complex microbiota and immune cells to allow further study into the effects of diet and related metabolites on the gut-immune ecosystem and their role in the prevention and development of metabolic diseases in humans.

Recreating the biological steps of viral infection on a cell-free bioelectronic platform to profile viral variants of concern.

Viral mutations frequently outpace technologies used to detect harmful variants. Given the continual emergence of SARS-CoV-2 variants, platforms that can identify the presence of a virus and its propensity for infection are needed. Our electronic biomembrane sensing platform recreates distinct SARS-CoV-2 host cell entry pathways and reports the progression of entry as electrical signals. We focus on two necessary entry processes mediated by the viral Spike protein: virus binding and membrane fusion, which can be distinguished electrically. We find that closely related variants of concern exhibit distinct fusion signatures that correlate with trends in cell-based infectivity assays, allowing us to report quantitative differences in their fusion characteristics and hence their infectivity potentials. We use SARS-CoV-2 as our prototype, but we anticipate that this platform can extend to other enveloped viruses and cell lines to quantifiably assess virus entry.

Simple dynamic cell culture system reduces recording noise in microelectrode array recordings.

Microelectrode arrays (MEAs) have applications in drug discovery, toxicology, and basic research. They measure the electrophysiological response of tissue cultures to quantify changes upon exposure to biochemical stimuli. Unfortunately, manual addition of chemicals introduces significant noise in the recordings. Here, we report a simple-to-fabricate fluidic system that addresses this issue. We show that cell cultures can be successfully established in the fluidic compartment under continuous flow conditions and that the addition of chemicals introduces minimal noise in the recordings. This dynamic cell culture system represents an improvement over traditional tissue culture wells used in MEAs, facilitating electrophysiology measurements.

In Vitro Models for Investigating Intestinal Host-Pathogen Interactions.

Infectious diseases are increasingly recognized as a major threat worldwide due to the rise of antimicrobial resistance and the emergence of novel pathogens. In vitro models that can adequately mimic in vivo gastrointestinal physiology are in high demand to elucidate mechanisms behind pathogen infectivity, and to aid the design of effective preventive and therapeutic interventions. There exists a trade-off between simple and high throughput models and those that are more complex and physiologically relevant. The complexity of the model used shall be guided by the biological question to be addressed. This review provides an overview of the structure and function of the intestine and the models that are developed to emulate this. Conventional models are discussed in addition to emerging models which employ engineering principles to equip them with necessary advanced monitoring capabilities for intestinal host-pathogen interrogation. Limitations of current models and future perspectives on the field are presented.

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.

A Conformable Organic Electronic Device for Monitoring Epithelial Integrity at the Air Liquid Interface.

Air liquid interfaced (ALI) epithelial barriers are essential for homeostatic functions such as nutrient transport and immunological protection. Dysfunction of such barriers are implicated in a variety of autoimmune and inflammatory disorders and, as such, sensors capable of monitoring barrier health are integral for disease modelling, diagnostics and drug screening applications. To date, gold-standard electrical methods for detecting barrier resistance require rigid electrodes bathed in an electrolyte, which limits compatibility with biological architectures and is non-physiological for ALI. This work presents a flexible all-planar electronic device capable of monitoring barrier formation and perturbations in human respiratory and intestinal cells at ALI. By interrogating patient samples with electrochemical impedance spectroscopy and simple equivalent circuit models, disease-specific and patient-specific signatures are uncovered. Device readouts are validated against commercially available chopstick electrodes and show greater conformability, sensitivity and biocompatibility. The effect of electrode size on sensing efficiency is investigated and a cut-off sensing area is established, which is one order of magnitude smaller than previously reported. This work provides the first steps in creating a physiologically relevant sensor capable of mapping local and real-time changes of epithelial barrier function at ALI, which will have broad applications in toxicology and drug screening applications.