Cell viability and cytotoxicity of inkjet-printed flexible organic electrodes on parylene C.

This study reports on the fabrication of biocompatible organic devices by means of inkjet printing with a novel combination of materials. The devices were fabricated on Parylene C (PaC), a biocompatible and flexible polymer substrate. The contact tracks were inkjet-printed using a silver nanoparticle ink, while the active sites were inkjet-printed using a poly (3,4ethylenedioxythiophene)/polystyrene sulfonate (PEDOT:PSS) solution. To insulate the final device, a polyimide ink was used to print a thick film, leaving small open windows upon the active sites. Electrical characterization of the final device revealed conductivities in the order of 103 and 102 S.cm-1 for Ag and PEDOT based inks, respectively. Cell adhesion assays performed with PC-12 cells after 96 h of culture, and B16F10 cells after 24 h of culture, demonstrated that the cells adhered on top of the inks and cell differentiation occurred, which indicates Polyimide and PEDOT:PSS inks are non-toxic to these cells. The results indicate that PaC, along with its surface-treated variants, is a potentially useful material for fabricating cell-based microdevices.

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.

Flexible switch matrix addressable electrode arrays with organic electrochemical transistor and pn diode technology.

Due to their effective ionic-to-electronic signal conversion and mechanical flexibility, organic neural implants hold considerable promise for biocompatible neural interfaces. Current approaches are, however, primarily limited to passive electrodes due to a lack of circuit components to realize complex active circuits at the front-end. Here, we introduce a p-n organic electrochemical diode using complementary p- and n-type conducting polymer films embedded in a 15-µm -diameter vertical stack. Leveraging the efficient motion of encapsulated cations inside this polymer stack and the opposite doping mechanisms of the constituent polymers, we demonstrate high current rectification ratios ([Formula: see text]) and fast switching speeds (230 µs). We integrate p-n organic electrochemical diodes with organic electrochemical transistors in the front-end pixel of a recording array. This configuration facilitates the access of organic electrochemical transistor output currents within a large network operating in the same electrolyte, while minimizing crosstalk from neighboring elements due to minimized reverse-biased leakage. Furthermore, we use these devices to fabricate time-division-multiplexed amplifier arrays. Lastly, we show that, when fabricated in a shank format, this technology enables the multiplexing of amplified local field potentials directly in the active recording pixel (26-µm diameter) in a minimally invasive form factor with shank cross-sectional dimensions of only 50×8 [Formula: see text].

Reconfiguration of organic electrochemical transistors for high-accuracy potentiometric sensing.

Organic electrochemical transistors have emerged as a promising alternative to traditional 2/3 electrode setups for sensing applications, offering in-situ transduction, electrochemical amplification, and noise reduction. Several of these devices are designed to detect potentiometric-derived signals. However, potentiometric sensing should be performed under open circuit potential conditions, allowing the system to reach thermodynamic equilibrium. This criterion is not met by conventional organic electrochemical transistors, where voltages or currents are directly applied to the sensing interface, that is, the gate electrode. In this work, we introduce an organic electrochemical transistor sensing configuration called the potentiometric­OECT (pOECT), which maintains the sensing electrode under open circuit potential conditions. The pOECT exhibits a higher response than the 2-electrode setup and offers greater accuracy, response, and stability compared to conventional organic electrochemical transistors. Additionally, it allows for the implementation of high-impedance electrodes as gate/sensing surfaces, all without compromising the overall device size.

SpyDirect: A Novel Biofunctionalization Method for High Stability and Longevity of Electronic Biosensors.

Electronic immunosensors are indispensable tools for diagnostics, particularly in scenarios demanding immediate results. Conventionally, these sensors rely on the chemical immobilization of antibodies onto electrodes. However, globular proteins tend to adsorb and unfold on these surfaces. Therefore, self-assembled monolayers (SAMs) of thiolated alkyl molecules are commonly used for indirect gold-antibody coupling. Here, a limitation associated with SAMs is revealed, wherein they curtail the longevity of protein sensors, particularly when integrated into the state-of-the-art transducer of organic bioelectronics-the organic electrochemical transistor. The SpyDirect method is introduced, generating an ultrahigh-density array of oriented nanobody receptors stably linked to the gold electrode without any SAMs. It is accomplished by directly coupling cysteine-terminated and orientation-optimized spyTag peptides, onto which nanobody-spyCatcher fusion proteins are autocatalytically attached, yielding a dense and uniform biorecognition layer. The structure-guided design optimizes the conformation and packing of flexibly tethered nanobodies. This biolayer enhances shelf-life and reduces background noise in various complex media. SpyDirect functionalization is faster and easier than SAM-based methods and does not necessitate organic solvents, rendering the sensors eco-friendly, accessible, and amenable to scalability. SpyDirect represents a broadly applicable biofunctionalization method for enhancing the cost-effectiveness, sustainability, and longevity of electronic biosensors, all without compromising sensitivity.

Interactions of Catalytic Enzymes with n-Type Polymers for High-Performance Metabolite Sensors.

The tight regulation of the glucose concentration in the body is crucial for balanced physiological function. We developed an electrochemical transistor comprising an n-type conjugated polymer film in contact with a catalytic enzyme for sensitive and selective glucose detection in bodily fluids. Despite the promise of these sensors, the property of the polymer that led to such high performance has remained unknown, with charge transport being the only characteristic under focus. Here, we studied the impact of the polymer chemical structure on film surface properties and enzyme adsorption behavior using a combination of physiochemical characterization methods and correlated our findings with the resulting sensor performance. We developed five n-type polymers bearing the same backbone with side chains differing in polarity and charge. We found that the nature of the side chains modulated the film surface properties, dictating the extent of interactions between the enzyme and the polymer film. Quartz crystal microbalance with dissipation monitoring studies showed that hydrophobic surfaces retained more enzymes in a densely packed arrangement, while hydrophilic surfaces captured fewer enzymes in a flattened conformation. X-ray photoelectron spectroscopy analysis of the surfaces revealed strong interactions of the enzyme with the glycolated side chains of the polymers, which improved for linear side chains compared to those for branched ones. We probed the alterations in the enzyme structure upon adsorption using circular dichroism, which suggested protein denaturation on hydrophobic surfaces. Our study concludes that a negatively charged, smooth, and hydrophilic film surface provides the best environment for enzyme adsorption with desired mass and conformation, maximizing the sensor performance. This knowledge will guide synthetic work aiming to establish close interactions between proteins and electronic materials, which is crucial for developing high-performance enzymatic metabolite biosensors and biocatalytic charge-conversion devices.

Photo-Chemical Stimulation of Neurons with Organic Semiconductors.

Recent advances in light-responsive materials enabled the development of devices that can wirelessly activate tissue with light. Here it is shown that solution-processed organic heterojunctions can stimulate the activity of primary neurons at low intensities of light via photochemical reactions. The p-type semiconducting polymer PDCBT and the n-type semiconducting small molecule ITIC (a non-fullerene acceptor) are coated on glass supports, forming a p-n junction with high photosensitivity. Patch clamp measurements show that low-intensity white light is converted into a cue that triggers action potentials in primary cortical neurons. The study shows that neat organic semiconducting p-n bilayers can exchange photogenerated charges with oxygen and other chemical compounds in cell culture conditions. Through several controlled experimental conditions, photo-capacitive, photo-thermal, and direct hydrogen peroxide effects on neural function are excluded, with photochemical delivery being the possible mechanism. The profound advantages of low-intensity photo-chemical intervention with neuron electrophysiology pave the way for developing wireless light-based therapy based on emerging organic semiconductors.

Organic Electronic Platform for Real-Time Phenotypic Screening of Extracellular-Vesicle-Driven Breast Cancer Metastasis.

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.

Investigation of the potential of biocalorimetry as a process analytical technology (PAT) tool for monitoring and control of Crabtree-negative yeast cultures.

Biological reaction calorimetry, also known as biocalorimetry, has led to extensive applications in monitoring and control of different bioprocesses. A simple real-time estimator for biomass and growth rate was formulated, based on in-line measured metabolic heat flow values. The performance of the estimator was tested in a unique bench-scale calorimeter (BioRC1), improved to a sensitivity range of 8 mW l(-1) in order to facilitate the monitoring of even weakly exothermic biochemical reactions. A proportional-integral feedback control strategy based on these estimators was designed and implemented to control the growth rate of Candida utilis, Kluyveromyces marxianus and Pichia pastoris by regulating an exponential substrate feed. Maintaining a particular specific growth rate throughout a culture is essential for reproducible product quality in industrial bioprocesses and therefore a key sequence for the step from quality by analysis to quality by design. The potential of biocalorimetry as a reliable biomass monitoring tool and as a key part of a robust control strategy for aerobic fed-batch cultures of Crabtree-negative yeast cells in defined growth medium was investigated. Presenting controller errors of less than 4% in the best cases, the approach paves the way for the development of a generally applicable process analytical technology platform for monitoring and control of microbial fed-batch cultures.

Biostack: Nontoxic Metabolite Detection from Live Tissue.

There is increasing demand for direct in situ metabolite monitoring from cell cultures and in vivo using implantable devices. Electrochemical biosensors are commonly preferred due to their low-cost, high sensitivity, and low complexity. Metabolite detection, however, in cultured cells or sensitive tissue is rarely shown. Commonly, glucose sensing occurs indirectly by measuring the concentration of hydrogen peroxide, which is a by-product of the conversion of glucose by glucose oxidase. However, continuous production of hydrogen peroxide in cell media with high glucose is toxic to adjacent cells or tissue. This challenge is overcome through a novel, stacked enzyme configuration. A primary enzyme is used to provide analyte sensitivity, along with a secondary enzyme which converts H2 O2 back to O2 . The secondary enzyme is functionalized as the outermost layer of the device. Thus, production of H2 O2 remains local to the sensor and its concentration in the extracellular environment does not increase. This "biostack" is integrated with organic electrochemical transistors to demonstrate sensors that monitor glucose concentration in cell cultures in situ. The "biostack" renders the sensors nontoxic for cells and provides highly sensitive and stable detection of metabolites.

Convection Driven Ultrarapid Protein Detection via Nanobody-Functionalized Organic Electrochemical Transistors.

Conventional biosensors rely on the diffusion-dominated transport of the target analyte to the sensor surface. Consequently, they require an incubation step that may take several hours to allow for the capture of analyte molecules by sensor biorecognition sites. This incubation step is a primary cause of long sample-to-result times. Here, alternating current electrothermal flow (ACET) is integrated in an organic electrochemical transistor (OECT)-based sensor to accelerate the device operation. ACET is applied to the gate electrode functionalized with nanobody-SpyCatcher fusion proteins. Using the SARS-CoV-2 spike protein in human saliva as an example target, it is shown that ACET enables protein recognition within only 2 min of sample exposure, supporting its use in clinical practice. The ACET integrated sensor exhibits better selectivity, higher sensitivity, and lower limit of detection than the equivalent sensor with diffusion-dominated operation. The performance of ACET integrated sensors is compared with two types of organic semiconductors in the channel and grounds for device-to-device variations are investigated. The results provide guidelines for the channel material choice in OECT-based biochemical sensors, and demonstrate that ACET integration substantially decreases the detection speed while increasing the sensitivity and selectivity of transistor-based sensors.

Muscle Fatigue Sensor Based on Ti3 C2 Tx MXene Hydrogel.

MXene-based hydrogels have received significant attention due to several promising properties that distinguish them from conventional hydrogels. In this study, it is shown that both strain and pH level can be exploited to tune the electronic and ionic transport in MXene-based hydrogel (M-hydrogel), which consists of MXene (Ti3 C2 Tx )-polyacrylic acid/polyvinyl alcohol hydrogel. In particular, the strain applied to the M-hydrogel changes MXene sheet orientation which leads to modulation of ionic transport within the M-hydrogel, due to strain-induced orientation of the surface charge-guided ionic pathway. Simultaneously, the reorientation of MXene sheets under the axial strain increases the electronic resistance of the M-hydrogel due to the loss of the percolative network of conductive MXene sheets during the stretching process. The iontronic characteristics of the M-hydrogel can thus be tuned by strain and pH, which allows using the M-hydrogel as a muscle fatigue sensor during exercise. A fully functional M-hydrogel is developed for real-time measurement of muscle fatigue during exercise and coupled it to a smartphone to provide a portable or wearable digital readout. This concept can be extended to other fields that require accurate analysis of constantly changing physical and chemical conditions, such as physiological changes in the human body.

Dual Mode Sensing of Binding and Blocking of Cancer Exosomes to Biomimetic Human Primary Stem Cell Surfaces.

Cancer-derived exosomes (cEXOs) facilitate transfer of information between tumor and human primary stromal cells, favoring cancer progression. Although the mechanisms used during this information exchange are still not completely understood, it is known that binding is the initial contact established between cEXOs and cells. Hence, studying binding and finding strategies to block it are of great therapeutic value. However, such studies are challenging for a variety of reasons, including the need for human primary cell culture, the difficulty in decoupling and isolating binding from internalization and cargo delivery, and the lack of techniques to detect these specific interactions. In this work, we created a supported biomimetic stem cell membrane incorporating membrane components from human primary adipose-derived stem cells (ADSCs). We formed the supported membrane on glass and on multielectrode arrays to offer the dual option of optical or electrical detection of cEXO binding to the membrane surface. Using our platform, we show that cEXOs bind to the stem cell membrane and that binding is blocked when an antibody to integrin ß1, a component of ADSC surface, is exposed to the membrane surface prior to cEXOs. To test the biological outcome of blocking this interaction, we first confirm that adding cEXOs to cultured ADSCs leads to the upregulation of vascular endothelial growth factor, a measure of proangiogenic activity. Next, when ADSCs are first blocked with anti-integrin ß1 and then exposed to cEXOs, the upregulation of proangiogenic activity and cell proliferation are significantly reduced. This biomimetic membrane platform is the first cell-free label-free in vitro platform for the recapitulation and study of cEXO binding to human primary stem cells with potential for therapeutic molecule screening as it is compatible with scale-up and multiplexing.

Rapid single-molecule detection of COVID-19 and MERS antigens via nanobody-functionalized organic electrochemical transistors.

The coronavirus disease 2019 (COVID-19) pandemic has highlighted the need for rapid and sensitive protein detection and quantification in simple and robust formats for widespread point-of-care applications. Here, we report on nanobody-functionalized organic electrochemical transistors with a modular architecture for the rapid quantification of single-molecule-to-nanomolar levels of specific antigens in complex bodily fluids. The sensors combine a solution-processable conjugated polymer in the transistor channel and high-density and orientation-controlled bioconjugation of nanobody-SpyCatcher fusion proteins on disposable gate electrodes. The devices provide results after 10 min of exposure to 5 µl of unprocessed samples, maintain high specificity and single-molecule sensitivity in human saliva and serum, and can be reprogrammed to detect any protein antigen if a corresponding specific nanobody is available. We used the sensors to detect green fluorescent protein, and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and Middle East respiratory syndrome coronavirus (MERS-CoV) spike proteins, and for the COVID-19 screening of unprocessed clinical nasopharyngeal swab and saliva samples with a wide range of viral loads.

Benchmarking the Performance of Electropolymerized Poly(3,4-ethylenedioxythiophene) Electrodes for Neural Interfacing.

The development of electronics adept at interfacing with the nervous system is an ever-growing effort, leading to discoveries in fundamental neuroscience applied in clinical setting. Highly capacitive and electrochemically stable electronic materials are paramount for these advances. A systematic study is presented where copolymers based on 3,4-ethylenedioxythiophene (EDOT) and its hydroxyl-terminated counterpart (EDOTOH) are electropolymerized in an aqueous solution in the presence of various counter anions and additives. Amongst the conducting materials developed, the copolymer p(EDOT-ran-EDOTOH) doped with perchlorate in the presence of ethylene glycol shows high specific capacitance (105 F g-1 ), and capacitance retention (85%) over 1000 galvanostatic charge-discharge cycles. A microelectrode array-based on this material is fabricated and primary cortical neurons are cultured therein for several days. The microelectrodes electrically stimulate targeted neuronal networks and record their activity with high signal-to-noise ratio. The stability of charge injection capacity of the material is validated via long-term pulsing experiments. While providing insights on the effect of additives and dopants on the electrochemical performance and operational stability of electropolymerized conducting polymers, this study highlights the importance of high capacitance accompanied with stability to achieve high performance electrodes for biological interfacing.

Visualizing the Solid-Liquid Interface of Conjugated Copolymer Films Using Fluorescent Liposomes.

Conjugated polymers are promising engineering tools for establishing bilateral electrical communication with living systems. The free energy of their films, the roughness, and charge density play major roles in determining their interactions with lipid bilayers, which form the membrane barrier around every living cell allowing for molecular exchange with the extracellular environment. In this work, we investigate lipid bilayer formation from synthetic lipid vesicles (liposomes) on a series of amphiphilic copolymer films based on naphthalene 1,4,5,8 tetracarboxylic diimide bithiophene (NDI-T2) backbone where the alkyl side chain is gradually exchanged for an ethylene glycol-based side chain. As the concentration of ethylene glycol in the composition changes, the surface energy of the films varies drastically, which, in turn, effects the interactions with liposomes. By imaging the interactions of fluorophore-labeled liposomes with these surfaces via a fluorescence microscope, we show that the films can be cast such that ethylene glycol-rich regions, which liposomes favor, are accumulated on the surface and extract information on the wettability behavior that has not been possible using other surface sensitive techniques. This approach uncovers the solid/liquid interface of a promising class of electron transporting conjugated polymer films and suggests synthetic strategies to maximize the number of lipid-polymer contacts for the formation of supported lipid bilayers.

Organic transistor platform with integrated microfluidics for in-line multi-parametric in vitro cell monitoring.

Future drug discovery and toxicology testing could benefit significantly from more predictive and multi-parametric readouts from in vitro models. Despite the recent advances in the field of microfluidics, and more recently organ-on-a-chip technology, there is still a high demand for real-time monitoring systems that can be readily embedded with microfluidics. In addition, multi-parametric monitoring is essential to improve the predictive quality of the data used to inform clinical studies that follow. Here we present a microfluidic platform integrated with in-line electronic sensors based on the organic electrochemical transistor. Our goals are two-fold, first to generate a platform to host cells in a more physiologically relevant environment (using physiologically relevant fluid shear stress (FSS)) and second to show efficient integration of multiple different methods for assessing cell morphology, differentiation, and integrity. These include optical imaging, impedance monitoring, metabolite sensing, and a wound-healing assay. We illustrate the versatility of this multi-parametric monitoring in giving us increased confidence to validate the improved differentiation of cells toward a physiological profile under FSS, thus yielding more accurate data when used to assess the effect of drugs or toxins. Overall, this platform will enable high-content screening for in vitro drug discovery and toxicology testing and bridges the existing gap in the integration of in-line sensors in microfluidic devices.

Polyelectrolyte Layer-by-Layer Assembly on Organic Electrochemical Transistors.

Oppositely charged polyelectrolyte multilayers (PEMs) were built up in a layer-by-layer (LbL) assembly on top of the conducting polymer channel of an organic electrochemical transistor (OECT), aiming to combine the advantages of well-established PEMs with a high performance electronic transducer. The multilayered film is a model system to investigate the impact of biofunctionalization on the operation of OECTs comprising a poly(3,4-ethylenedioxythiophene) polystyrenesulfonate (PEDOT:PSS) film as the electrically active layer. Understanding the mechanism of ion injection into the channel that is in direct contact with charged polymer films provides useful insights for novel biosensing applications such as nucleic acid sensing. Moreover, LbL is demonstrated to be a versatile electrode modification tool enabling tailored surface features in terms of thickness, softness, roughness, and charge. LbL assemblies built up on top of conducting polymers will aid the design of new bioelectronic platforms for drug delivery, tissue engineering, and medical diagnostics.