Dynamic monitoring of a 3D-printed airway tissue model using an organic electrochemical transistor.

Assessing the transepithelial resistance to ion flow in the presence of an electric field enables the evaluation of the integrity of the epithelial cell layer. In this study, we introduce an organic electrochemical transistor (OECT) interfaced with a 3D living tissue, designed to monitor the electrical resistance of cellular barriers in real-time. We have developed a non-invasive, tissue-sensing platform by integrating an inkjet-printed large-area OECT with a 3D-bioprinted multilayered airway tissue. This unique configuration enables the evaluation of epithelial barrier integrity through the dynamic response capabilities of the OECT. Our system effectively tracks the formation and integrity of 3D-printed airway tissues in both liquid-liquid and air-liquid interface culture environments. Furthermore, we successfully quantified the degradation of barrier function due to influenza A (H1N1) viral infection and the dose-dependent efficacy of oseltamivir (Tamiflu®) in mitigating this degradation. The tissue-electronic platform offers a non-invasive and label-free method for real-time monitoring of 3D artificial tissue barriers, without disturbing the cellular biology. It holds the potential for further applications in monitoring the structures and functions of 3D tissues and organs, significantly contributing to the advancement of personalized medicine.

Reaching across the divide: materials scientists interfacing with biologists.

Scientific research is becoming increasingly interdisciplinary and poses new challenges, undertakings, and prospects. In this article, we discuss the various aspects of interdisciplinarity in the developing field of organic bioelectronics. The authors represent two different fields, namely, biochemistry and materials science, and discuss their perspectives on working together in a scientifically diverse research environment. We outline today's challenges based on personal experiences and present possible solutions and hopeful opportunities for the future.

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.

Co-transcriptional production of programmable RNA condensates and synthetic organelles.

Condensation of RNA and proteins is central to cellular functions, and the ability to program it would be valuable in synthetic biology and synthetic cell science. Here we introduce a modular platform for engineering synthetic RNA condensates from tailor-made, branched RNA nanostructures that fold and assemble co-transcriptionally. Up to three orthogonal condensates can form simultaneously and selectively accumulate fluorophores through embedded fluorescent light-up aptamers. The RNA condensates can be expressed within synthetic cells to produce membrane-less organelles with a controlled number and relative size, and showing the ability to capture proteins using selective protein-binding aptamers. The affinity between otherwise orthogonal nanostructures can be modulated by introducing dedicated linker constructs, enabling the production of bi-phasic RNA condensates with a prescribed degree of interphase mixing and diverse morphologies. The in situ expression of programmable RNA condensates could underpin the spatial organization of functionalities in both biological and synthetic cells.

Stretchable Device for Simultaneous Measurements of Contractility and Electrophysiology of Neuromuscular Tissue in the Gastrointestinal Tract.

Devices interfacing with biological tissues can provide valuable insights into function, disease, and metabolism through electrical and mechanical signals. However, certain neuromuscular tissues, like those in the gastrointestinal tract, undergo significant strains of up to 40%. Conventional inextensible devices cannot capture the dynamic responses in these tissues. This study introduces electrodes made from poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS) and polydimethylsiloxane (PDMS) that enable simultaneous monitoring of electrical and mechanical responses of gut tissue. The soft PDMS layers conform to tissue surfaces during gastrointestinal movement. Dopants, including Capstone FS-30 and polyethylene glycol, are explored to enhance the conductivity, electrical sensitivity to strain, and stability of the PEDOT:PSS. The devices are fabricated using shadow masks and solution-processing techniques, providing a faster and simpler process than traditional clean-room-based lithography. Tested on ex vivo mouse colon and human stomach, the device recorded voltage changes of up to 300 µV during contraction and distension consistent with muscle activity, while simultaneously recording resistance changes of up to 150% due to mechanical strain. These devices detect and respond to chemical stimulants and blockers, and can induce contractions through electrical stimulation. They hold great potential for studying and treating complex disorders like irritable bowel syndrome and gastroparesis.

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.

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.

Modelling bronchial epithelial-fibroblast cross-talk in idiopathic pulmonary fibrosis (IPF) using a human-derived in vitro air liquid interface (ALI) culture.

Idiopathic Pulmonary Fibrosis (IPF) is a devastating form of respiratory disease with a life expectancy of 3-4 years. Inflammation, epithelial injury and myofibroblast proliferation have been implicated in disease initiation and, recently, epithelial-fibroblastic crosstalk has been identified as a central driver. However, the ability to interrogate this crosstalk is limited due to the absence of in vitro models that mimic physiological conditions. To investigate IPF dysregulated cross-talk, primary normal human bronchial epithelial (NHBE) cells and primary normal human lung fibroblasts (NHLF) or diseased human lung fibroblasts (DHLF) from IPF patients, were co-cultured in direct contact at the air-liquid interface (ALI). Intercellular crosstalk was assessed by comparing cellular phenotypes of co-cultures to respective monocultures, through optical, biomolecular and electrical methods. A co-culture-dependent decrease in epithelium thickness, basal cell mRNA (P63, KRT5) and an increase in transepithelial electrical resistance (TEER) was observed. This effect was significantly enhanced in DHLF co-cultures and lead to the induction of epithelial to mesenchymal transition (EMT) and increased mRNA expression of TGFß-2, ZO-1 and DN12. When stimulated with exogenous TGFß, NHBE and NHLF monocultures showed a significant upregulation of EMT (COL1A1, FN1, VIM, ASMA) and senescence (P21) markers, respectively. In contrast, direct NHLF/NHBE co-culture indicated a protective role of epithelial-fibroblastic cross-talk against TGFß-induced EMT, fibroblast-to-myofibroblast transition (FMT) and inflammatory cytokine release (IL-6, IL-8, IL-13, IL-1ß, TNF-α). DHLF co-cultures showed no significant phenotypic transition upon stimulation, likely due to the constitutively high expression of TGFß isoforms prior to any exogenous stimulation. The model developed provides an alternative method to generate IPF-related bronchial epithelial phenotypes in vitro, through the direct co-culture of human lung fibroblasts with NHBEs. These findings highlight the importance of fibroblast TGFß signaling in EMT but that monocultures give rise to differential responses compared to co-cultures, when exposed to this pro-inflammatory stimulus. This holds implications for any translation conclusions drawn from monoculture studies and is an important step in development of more biomimetic models of IPF. In summary, we believe this in vitro system to study fibroblast-epithelial crosstalk, within the context of IPF, provides a platform which will aid in the identification and validation of novel targets.

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.

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.

Microelectrode Arrays Measure Blocking of Voltage-Gated Calcium Ion Channels on Supported Lipid Bilayers Derived from Primary Neurons.

Drug studies targeting neuronal ion channels are crucial to understand neuronal function and develop therapies for neurological diseases. The traditional method to study neuronal ion-channel activities heavily relies on the whole-cell patch clamp as the industry standard. However, this technique is both technically challenging and labour-intensive, while involving the complexity of keeping cells alive with low throughput. Therefore, the shortcomings are limiting the efficiency of ion-channel-related neuroscience research and drug testing. Here, this work reports a new system of integrating neuron membranes with organic microelectrode arrays (OMEAs) for ion-channel-related drug studies. This work demonstrates that the supported lipid bilayers (SLBs) derived from both neuron-like (neuroblastoma) cells and primary neurons are integrated with OMEAs for the first time. The increased expression of voltage-gated calcium (CaV) ion channels on differentiated SH-SY5Y SLBs  compared to non-differentiated ones is sensed electrically. Also, dose-response of the CaV ion-channel blocking effect on primary cortical neuronal SLBs from rats is monitored. The dose range causing ion channel blocking is comparable to literature. This system overcomes the major challenges from traditional methods (e.g., patch clamp) and showcases an easy-to-test, rapid, ultra-sensitive, cell-free, and high-throughput platform to monitor dose-dependent ion-channel blocking effects on native neuronal membranes.

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.

Protein Language Models Uncover Carbohydrate-Active Enzyme Function in Metagenomics.

In metagenomics, the pool of uncharacterized microbial enzymes presents a challenge for functional annotation. Among these, carbohydrate-active enzymes (CAZymes) stand out due to their pivotal roles in various biological processes related to host health and nutrition. Here, we present CAZyLingua, the first tool that harnesses protein language model embeddings to build a deep learning framework that facilitates the annotation of CAZymes in metagenomic datasets. Our benchmarking results showed on average a higher F1 score (reflecting an average of precision and recall) on the annotated genomes of Bacteroides thetaiotaomicron, Eggerthella lenta and Ruminococcus gnavus compared to the traditional sequence homology-based method in dbCAN2. We applied our tool to a paired mother/infant longitudinal dataset and revealed unannotated CAZymes linked to microbial development during infancy. When applied to metagenomic datasets derived from patients affected by fibrosis-prone diseases such as Crohn's disease and IgG4-related disease, CAZyLingua uncovered CAZymes associated with disease and healthy states. In each of these metagenomic catalogs, CAZyLingua discovered new annotations that were previously overlooked by traditional sequence homology tools. Overall, the deep learning model CAZyLingua can be applied in combination with existing tools to unravel intricate CAZyme evolutionary profiles and patterns, contributing to a more comprehensive understanding of microbial metabolic dynamics.

Effects of Processing-Induced Contamination on Organic Electronic Devices.

Organic semiconductors are a family of pi-conjugated compounds used in many applications, such as displays, bioelectronics, and thermoelectrics. However, their susceptibility to processing-induced contamination is not well understood. Here, it is shown that many organic electronic devices reported so far may have been unintentionally contaminated, thus affecting their performance, water uptake, and thin film properties. Nuclear magnetic resonance spectroscopy is used to detect and quantify contaminants originating from the glovebox atmosphere and common laboratory consumables used during device fabrication. Importantly, this in-depth understanding of the sources of contamination allows the establishment of clean fabrication protocols, and the fabrication of organic field effect transistors (OFETs) with improved performance and stability. This study highlights the role of unintentional contaminants in organic electronic devices, and demonstrates that certain stringent processing conditions need to be met to avoid scientific misinterpretation, ensure device reproducibility, and facilitate performance stability. The experimental procedures and conditions used herein are typical of those used by many groups in the field of solution-processed organic semiconductors. Therefore, the insights gained into the effects of contamination are likely to be broadly applicable to studies, not just of OFETs, but also of other devices based on these materials.

Activity-enhanced DNAzyme for design of label-free copper(II) biosensor.

Metal ion-driven, DNA-cleaving DNAzymes are characterised by high selectivity and specificity. However, their use for metal ion sensing remains largely unexplored due to long reaction times and poor reaction yields relative to RNA-cleaving DNAzymes and other sensing strategies. Herein we present a study demonstrating a significant rate enhancement of a copper-selective DNA cleaving DNAzyme by both polydopamine (PDA) and gold (Au) nanoparticles (NPs). PDA NPs enhance the reaction through the production of hydrogen peroxide, while for AuNPs the enhancement is aided by the presence of citrate surface moeities, both of which drive the oxidative cleavage of the substrate. A 50-fold enhancement for PDA NPs makes the combination of PDA and DNAzyme suitable for a practical application as a sensitive biosensor for Cu(II) ions. Using DNAzyme deposition onto a gold electrode followed by Polydopamine Assisted DNA Immobilisation (PADI), we achieve a cost-effective, label-free and fast (within 15 min) electrochemical biosensor with a limit of detection of 180 nmol (11 ppm), thus opening a route for the rational design of a new generation of hybrid DNAzyme-based biosensors.

3D organic bioelectronics for electrical monitoring of human adult stem cells.

Three-dimensional in vitro stem cell models have enabled a fundamental understanding of cues that direct stem cell fate. While sophisticated 3D tissues can be generated, technology that can accurately monitor these complex models in a high-throughput and non-invasive manner is not well adapted. Here we show the development of 3D bioelectronic devices based on the electroactive polymer poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate)-(PEDOT:PSS) and their use for non-invasive, electrical monitoring of stem cell growth. We show that the electrical, mechanical and wetting properties as well as the pore size/architecture of 3D PEDOT:PSS scaffolds can be fine-tuned simply by changing the processing crosslinker additive. We present a comprehensive characterization of both 2D PEDOT:PSS thin films of controlled thicknesses, and 3D porous PEDOT:PSS structures made by the freeze-drying technique. By slicing the bulky scaffolds we generate homogeneous, porous 250 µm thick PEDOT:PSS slices, constituting biocompatible 3D constructs able to support stem cell cultures. These multifunctional slices are attached on indium-tin oxide substrates (ITO) with the help of an electrically active adhesion layer, enabling 3D bioelectronic devices with a characteristic and reproducible, frequency dependent impedance response. This response changes drastically when human adipose derived stem cells (hADSCs) grow within the porous PEDOT:PSS network as revealed by fluorescence microscopy. The increase of cell population within the PEDOT:PSS porous network impedes the charge flow at the interface between PEDOT:PSS and ITO, enabling the interface resistance (R1) to be used as a figure of merit to monitor the proliferation of stem cells. The non-invasive monitoring of stem cell growth allows for the subsequent differentiation 3D stem cell cultures into neuron like cells, as verified by immunofluorescence and RT-qPCR measurements. The strategy of controlling important properties of 3D PEDOT:PSS structures simply by altering processing parameters can be applied for development of a number of stem cell in vitro models as well as stem cell differentiation pathways. We believe the results presented here will advance 3D bioelectronic technology for both fundamental understanding of in vitro stem cell cultures as well as the development of personalized therapies.

Capture and Release of Cancer Cells Through Smart Bioelectronics.

Noninvasive collection of target cells such as circulating tumor cells (CTCs) is crucial for biology and medicine research. Conventional methods of cell collection are often complex, requiring either size-dependent sorting or invasive enzymatic reactions. Here, we show the development of a functional polymer film, which combines the thermoresponsive poly(N-isopropylacrylamide) and the conducting poly(3,4-ethylenedioxythiopene)/poly(styrene sulfonate), and its use for the capture and release of CTCs. When coated onto microfabricated gold electrodes, the proposed polymer films are capable of noninvasively capturing and controllably releasing cells while, at the same time, monitoring these processes with conventional electrical measurements.

Tissue-Engineered Cochlear Fibrosis Model Links Complex Impedance to Fibrosis Formation for Cochlear Implant Patients.

Cochlear implants are a life-changing technology for those with severe sensorineural hearing loss, partially restoring hearing through direct electrical stimulation of the auditory nerve. However, they are known to elicit an immune response resulting in fibrotic tissue formation in the cochlea that is linked to residual hearing loss and suboptimal outcomes. Intracochlear fibrosis is difficult to track without postmortem histology, and no specific electrical marker for fibrosis exists. In this study, a tissue-engineered model of cochlear fibrosis is developed following implant placement to examine the electrical characteristics associated with fibrotic tissue formation around electrodes. The model is characterized using electrochemical impedance spectroscopy and an increase in the resistance and a decrease in capacitance of the tissue using a representative circuit are found. This result informs a new marker of fibrosis progression over time that is extractable from voltage waveform responses, which can be directly measured in cochlear implant patients. This marker is tested in a small sample size of recently implanted cochlear implant patients, showing a significant increase over two postoperative timepoints. Using this system, complex impedance is demonstrated as a marker of fibrosis progression that is directly measurable from cochlear implants to enable real-time tracking of fibrosis formation in patients, creating opportunities for earlier treatment intervention to improve cochlear implant efficacy.