Single-Step Functionalization Strategy of Graphene Microtransistor Array with Chemically Modified Aptamers for Biosensing Applications.

Graphene solution-gated field-effect transistors (gSGFETs) offer high potential for chemical and biochemical sensing applications. Among the current trends to improve this technology, the functionalization processes are gaining relevance for its crucial impact on biosensing performance. Previous efforts are focused on simplifying the attachment procedure from standard multi-step to single-step strategies, but they still suffer from overreaction, and impurity issues and are limited to a particular ligand. Herein, a novel strategy for single-step immobilization of chemically modified aptamers with fluorenylmethyl and acridine moieties, based on a straightforward synthetic route to overcome the aforementioned limitations is presented. This approach is benchmarked versus a standard multi-step strategy using thrombin as detection model. In order to assess the reliability of the functionalization strategies 48-gSGFETs arrays are employed to acquire large datasets with multiple replicas. Graphene surface characterization demonstrates robust and higher efficiency in the chemical coupling of the aptamers with the single-step strategy, while the electrical response evaluation validates the sensing capability, allowing to implement different alternatives for data analysis and reduce the sensing variability. In this work, a new tool capable of overcome the functionalization challenges of graphene surfaces is provided, paving the way toward the standardization of gSGFETs for biosensing purposes.

Switchless Multiplexing of Graphene Active Sensor Arrays for Brain Mapping.

Sensor arrays used to detect electrophysiological signals from the brain are paramount in neuroscience. However, the number of sensors that can be interfaced with macroscopic data acquisition systems currently limits their bandwidth. This bottleneck originates in the fact that, typically, sensors are addressed individually, requiring a connection for each of them. Herein, we present the concept of frequency-division multiplexing (FDM) of neural signals by graphene sensors. We demonstrate the high performance of graphene transistors as mixers to perform amplitude modulation (AM) of neural signals in situ, which is used to transmit multiple signals through a shared metal line. This technology eliminates the need for switches, remarkably simplifying the technical complexity of state-of-the-art multiplexed neural probes. Besides, the scalability of FDM graphene neural probes has been thoroughly evaluated and their sensitivity demonstrated in vivo. Using this technology, we envision a new generation of high-count conformal neural probes for high bandwidth brain machine interfaces.

Distortion-Free Sensing of Neural Activity Using Graphene Transistors.

Graphene solution-gated field-effect transistors (g-SGFETs) are promising sensing devices to transduce electrochemical potential signals in an electrolyte bath. However, distortion mechanisms in g-SGFET, which can affect signals of large amplitude or high frequency, have not been evaluated. Here, a detailed characterization and modeling of the harmonic distortion and non-ideal frequency response in g-SGFETs is presented. This accurate description of the input-output relation of the g-SGFETs allows to define the voltage- and frequency-dependent transfer functions, which can be used to correct distortions in the transduced signals. The effect of signal distortion and its subsequent calibration are shown for different types of electrophysiological signals, spanning from large amplitude and low frequency cortical spreading depression events to low amplitude and high frequency action potentials. The thorough description of the distortion mechanisms presented in this article demonstrates that g-SGFETs can be used as distortion-free signal transducers not only for neural sensing, but also for a broader range of applications in which g-SGFET sensors are used.

High-resolution mapping of infraslow cortical brain activity enabled by graphene microtransistors.

Recording infraslow brain signals (<0.1 Hz) with microelectrodes is severely hampered by current microelectrode materials, primarily due to limitations resulting from voltage drift and high electrode impedance. Hence, most recording systems include high-pass filters that solve saturation issues but come hand in hand with loss of physiological and pathological information. In this work, we use flexible epicortical and intracortical arrays of graphene solution-gated field-effect transistors (gSGFETs) to map cortical spreading depression in rats and demonstrate that gSGFETs are able to record, with high fidelity, infraslow signals together with signals in the typical local field potential bandwidth. The wide recording bandwidth results from the direct field-effect coupling of the active transistor, in contrast to standard passive electrodes, as well as from the electrochemical inertness of graphene. Taking advantage of such functionality, we envision broad applications of gSGFET technology for monitoring infraslow brain activity both in research and in the clinic.

Stable Full-Inkjet-Printed Solid-State Ag/AgCl Reference Electrode.

With a growing demand for the availability of inexpensive, simple, and rapid prototyped devices, the prospect of miniaturization of the reference electrodes using printing techniques becomes promising. A stable and reusable full-inkjet-printed solid-state reference electrode (IPRE) was developed. The reference electrode was fully produced by consecutive inkjet printing of several layers. Ag ink was printed and chlorinated by NaClO printing, forming a Ag/AgCl pseudoreference electrode. Then a surface protection by printing a Cl--saturated polyvinyl butyral membrane finally gave a reference electrode that demonstrated an outstanding performance comparable to commercial ones. This full inkjet printing fabrication strategy will improve the viability of producing low-cost miniaturized reference electrodes with interest in many electrochemical sensor-dependent areas.

Bistability, Causality, and Complexity in Cortical Networks: An In Vitro Perturbational Study.

Measuring the spatiotemporal complexity of cortical responses to direct perturbations provides a reliable index of the brain's capacity for consciousness in humans under both physiological and pathological conditions. Upon loss of consciousness, the complex pattern of causal interactions observed during wakefulness collapses into a stereotypical slow wave, suggesting that cortical bistability may play a role. Bistability is mainly expressed in the form of slow oscillations, a default pattern of activity that emerges from cortical networks in conditions of functional or anatomical disconnection. Here, we employ an in vitro model to understand the relationship between bistability and complexity in cortical circuits. We adapted the perturbational complexity index applied in humans to electrically stimulated cortical slices under different neuromodulatory conditions. At this microscale level, we demonstrate that perturbational complexity can be effectively modulated by pharmacological reduction of bistability and, albeit to a lesser extent, by enhancement of excitability, providing mechanistic insights into the macroscale measurements performed in humans.

A Minimally Invasive Microsensor Specially Designed for Simultaneous Dissolved Oxygen and pH Biofilm Profiling.

A novel sensing device for simultaneous dissolved oxygen (DO) and pH monitoring specially designed for biofilm profiling is presented in this work. This device enabled the recording of instantaneous DO and pH dynamic profiles within biofilms, improving the tools available for the study and the characterization of biological systems. The microsensor consisted of two parallel arrays of microelectrodes. Microelectrodes used for DO sensing were bare gold electrodes, while microelectrodes used for pH sensing were platinum-based electrodes modified using electrodeposited iridium oxide. The device was fabricated with a polyimide (Kapton®) film of 127 µm as a substrate for minimizing the damage caused on the biofilm structure during its insertion. The electrodes were covered with a Nafion® layer to increase sensor stability and repeatability and to avoid electrode surface fouling. DO microelectrodes showed a linear response in the range 0-8 mg L-1, a detection limit of 0.05 mg L-1, and a sensitivity of 2.06 nA L mg-1. pH electrodes showed a linear super-Nernstian response (74.2 ± 0.7 mV/pH unit) in a wide pH range (pH 4-9). The multi-analyte sensor array was validated in a flat plate bioreactor where simultaneous and instantaneous pH and DO profiles within a sulfide oxidizing biofilm were recorded. The electrodes spatial resolution, the monitoring sensitivity, and the minimally invasive features exhibited by the proposed microsensor improved biofilm monitoring performance, enabling the quantification of mass transfer resistances and the assessment of biological activity.

A perfusion chamber for monitoring transepithelial NaCl transport in an in vitro model of the renal tubule.

Transepithelial electrical measurements in the renal tubule have provided a better understanding of how kidney regulates electrolyte and water homeostasis through the reabsorption of molecules and ions (e.g., H2 O and NaCl). While experiments and measurement techniques using native tissue are difficult to prepare and to reproduce, cell cultures conducted largely with the Ussing chamber lack the effect of fluid shear stress which is a key physiological stimulus in the renal tubule. To overcome these limitations, we present a modular perfusion chamber for long-term culture of renal epithelial cells under flow that allows the continuous and simultaneous monitoring of both transepithelial electrical parameters and transepithelial NaCl transport. The latter is obtained from electrical conductivity measurements since Na+ and Cl- are the ions that contribute most to the electrical conductivity of a standard physiological solution. The system was validated with epithelial monolayers of raTAL and NRK-52E cells that were characterized electrophysiologically for 5 days under different flow conditions (i.e., apical perfusion, basal, or both). In addition, apical to basal chemical gradients of NaCl (140/70 and 70/140 mM) were imposed in order to demonstrate the feasibility of this methodology for quantifying and monitoring in real time the transepithelial reabsorption of NaCl, which is a primary function of the renal tubule.

Resemblance of the human liver sinusoid in a fluidic device with biomedical and pharmaceutical applications.

Maintenance of the complex phenotype of primary hepatocytes in vitro represents a limitation for developing liver support systems and reliable tools for biomedical research and drug screening. We herein aimed at developing a biosystem able to preserve human and rodent hepatocytes phenotype in vitro based on the main characteristics of the liver sinusoid: unique cellular architecture, endothelial biodynamic stimulation, and parenchymal zonation. Primary hepatocytes and liver sinusoidal endothelial cells (LSEC) were isolated from control and cirrhotic human or control rat livers and cultured in conventional in vitro platforms or within our liver-resembling device. Hepatocytes phenotype, function, and response to hepatotoxic drugs were analyzed. Results evidenced that mimicking the in vivo sinusoidal environment within our biosystem, primary human and rat hepatocytes cocultured with functional LSEC maintained morphology and showed high albumin and urea production, enhanced cytochrome P450 family 3 subfamily A member 4 (CYP3A4) activity, and maintained expression of hepatocyte nuclear factor 4 alpha (hnf4α) and transporters, showing delayed hepatocyte dedifferentiation. In addition, differentiated hepatocytes cultured within this liver-resembling device responded to acute treatment with known hepatotoxic drugs significantly different from those seen in conventional culture platforms. In conclusion, this study describes a new bioengineered device that mimics the human sinusoid in vitro, representing a novel method to study liver diseases and toxicology.

Biofilm dynamics characterization using a novel DO-MEA sensor: mass transport and biokinetics.

Biodegradation process modeling is an essential tool for the optimization of biotechnologies related to gaseous pollutant treatment. In these technologies, the predominant role of biofilm, particularly under conditions of no mass transfer limitations, results in a need to determine what processes are occurring within the same. By measuring the interior of the biofilms, an increased knowledge of mass transport and biodegradation processes may be attained. This information is useful in order to develop more reliable models that take biofilm heterogeneity into account. In this study, a new methodology, based on a novel dissolved oxygen (DO) and mass transport microelectronic array (MEA) sensor, is presented in order to characterize a biofilm. Utilizing the MEA sensor, designed to obtain DO and diffusivity profiles with a single measurement, it was possible to obtain distributions of oxygen diffusivity and biokinetic parameters along a biofilm grown in a flat plate bioreactor (FPB). The results obtained for oxygen diffusivity, estimated from oxygenation profiles and direct measurements, revealed that changes in its distribution were reduced when increasing the liquid flow rate. It was also possible to observe the effect of biofilm heterogeneity through biokinetic parameters, estimated using the DO profiles. Biokinetic parameters, including maximum specific growth rate, the Monod half-saturation coefficient of oxygen, and the maintenance coefficient for oxygen which showed a marked variation across the biofilm, suggest that a tool that considers the heterogeneity of biofilms is essential for the optimization of biotechnologies.

New trends in quantitative assessment of the corneal barrier function.

The cornea is a very particular tissue due to its transparency and its barrier function as it has to resist against the daily insults of the external environment. In addition, maintenance of this barrier function is of crucial importance to ensure a correct corneal homeostasis. Here, the corneal epithelial permeability has been assessed in vivo by means of non-invasive tetrapolar impedance measurements, taking advantage of the huge impact of the ion fluxes in the passive electrical properties of living tissues. This has been possible by using a flexible sensor based in SU-8 photoresist. In this work, a further analysis focused on the validation of the presented sensor is performed by monitoring the healing process of corneas that were previously wounded. The obtained impedance measurements have been compared with the damaged area observed in corneal fluorescein staining images. The successful results confirm the feasibility of this novel method, as it represents a more sensitive in vivo and non-invasive test to assess low alterations of the epithelial permeability. Then, it could be used as an excellent complement to the fluorescein staining image evaluation.

A 3D bioprinted hydrogel gut-on-chip with integrated electrodes for transepithelial electrical resistance (TEER) measurements.

Conventional gut-on-chip (GOC) models typically represent the epithelial layer of the gut tissue, neglecting other important components such as the stromal compartment and the extracellular matrix (ECM) that play crucial roles in maintaining intestinal barrier integrity and function. These models often employ hard, flat porous membranes for cell culture, thus failing to recapitulate the soft environment and complex 3D architecture of the intestinal mucosa. Alternatively, hydrogels have been recently introduced in GOCs as ECM analogs to support the co-culture of intestinal cells inin vivo-like configurations, and thus opening new opportunities in the organ-on-chip field. In this work, we present an innovative GOC device that includes a 3D bioprinted hydrogel channel replicating the intestinal villi architecture containing both the epithelial and stromal compartments of the gut mucosa. The bioprinted hydrogels successfully support both the encapsulation of fibroblasts and their co-culture with intestinal epithelial cells under physiological flow conditions. Moreover, we successfully integrated electrodes into the microfluidic system to monitor the barrier formation in real time via transepithelial electrical resistance measurements.

Ethanol Solvation of Polymer Residues in Graphene Solution-Gated Field Effect Transistors.

The persistence of photoresist residues from microfabrication procedures causes significant obstacles in the technological advancement of graphene-based electronic devices. These residues induce undesired chemical doping effects, diminish carrier mobility, and deteriorate the signal-to-noise ratio, making them critical in certain contexts, including sensing and electrical recording applications. In graphene solution-gated field-effect transistors (gSGFETs), the presence of polymer contaminants makes it difficult to perform precise electrical measurements, introducing response variability and calibration challenges. Given the absence of viable short to midterm alternatives to polymer-intensive microfabrication techniques, a postpatterning treatment involving THF and ethanol solvents was evaluated, with ethanol being the most effective, environmentally sustainable, and safe method for residue removal. Employing a comprehensive analysis with XPS, AFM, and Raman spectroscopy, together with electrical characterization, we investigated the influence of residual polymers on graphene surface properties and transistor functionality. Ethanol treatment exhibited a pronounced enhancement in gSGFET performance, as evidenced by a shift in the charge neutrality point and reduced dispersion. This systematic cleaning methodology holds the potential to improve the reproducibility and precision in the manufacturing of graphene devices. Particularly, by using ethanol for residue removal, we align our methodology with the principles of green chemistry, minimizing environmental impact while advancing diverse graphene technology applications.

Organ-on-a-chip with integrated semitransparent organic electrodes for barrier function monitoring.

Organs-on-a-chip (OoC) are cell culture platforms that replicate key functional units of tissues in vitro. Barrier integrity and permeability evaluation are of utmost importance when studying barrier-forming tissues. Impedance spectroscopy is a powerful tool and is widely used to monitor barrier permeability and integrity in real-time. However, data comparison across devices is misleading due to the generation of a non-homogenous field across the tissue barrier, making impedance data normalization very challenging. In this work, we address this issue by integrating PEDOT:PSS electrodes for barrier function monitoring with impedance spectroscopy. The semitransparent PEDOT:PSS electrodes cover the entire cell culture membrane providing a homogenous electric field across the entire membrane making the cell culture area equally accountable to the measured impedance. To the best of our knowledge, PEDOT:PSS has never been used solely to monitor the impedance of cellular barriers while enabling optical inspection in the OoC. The performance of the device is demonstrated by lining the device with intestinal cells where we monitored barrier formation under flow conditions, as well as barrier disruption and recovery under exposure to a permeability enhancer. The barrier tightness and integrity, and the intercellular cleft have been evaluated by analyzing the full impedance spectrum. Furthermore, the device is autoclavable paving the way toward more sustainable OoC options.

Correction: Organ-on-a-chip with integrated semitransparent organic electrodes for barrier function monitoring.

Correction for 'Organ-on-a-chip with integrated semitransparent organic electrodes for barrier function monitoring' by Denise Marrero et al., Lab Chip, 2023, https://doi.org/10.1039/d2lc01097f.

Covalent functionalisation controlled by molecular design for the aptameric recognition of serotonin in graphene-based field-effect transistors.

In the last decade, solution-gated graphene field effect transistors (GFETs) showed their versatility in the development of a miniaturized multiplexed platform for electrophysiological recordings and sensing. Due to their working mechanism, the surface functionalisation and immobilisation of receptors are pivotal to ensure the proper functioning of devices. Herein, we present a controlled covalent functionalisation strategy based on molecular design and electrochemical triggering, which provide a monolayer-like functionalisation of micro-GFET arrays retaining the electronic properties of graphenes. The functionalisation layer as a receptor was then employed as the linker for serotonin aptamer conjugation. The micro-GFET arrays display sensitivity toward the target analyte in the micromolar range in a physiological buffer (PBS 10 mM). The sensor allows the in-flow real-time monitoring of serotonin transient concentrations with fast and reversible responses.

Increased sinusoidal pressure impairs liver endothelial mechanosensing, uncovering novel biomarkers of portal hypertension.

Background & Aims: Portal hypertension (PH) is a frequent and severe clinical syndrome associated with chronic liver disease. Considering the mechanobiological effects of hydrostatic pressure and shear stress on endothelial cells, we hypothesised that PH might influence the phenotype of liver sinusoidal endothelial cells (LSECs) during disease progression. The aim of this study was to investigate the effects of increased hydrodynamic pressure on LSECs and to identify endothelial-derived biomarkers of PH. Methods: Primary LSECs were cultured under normal or increased hydrodynamic pressure within a pathophysiological range (1 vs. 12 mmHg) using a microfluidic liver-on-a-chip device. RNA sequencing was used to identify pressure-sensitive genes, which were validated in liver biopsies from two independent cohorts of patients with chronic liver disease with PH (n = 73) and participants without PH (n = 23). Biomarker discovery was performed in two additional independent cohorts of 104 patients with PH and 18 patients without PH. Results: Transcriptomic analysis revealed marked deleterious effect of pathological pressure in LSECs and identified chromobox 7 (CBX7) as a key transcription factor diminished by pressure. Hepatic CBX7 downregulation was validated in patients with PH and significantly correlated with hepatic venous pressure gradient. MicroRNA 181a-5p was identified as pressure-induced upstream regulator of CBX7. Two downstream targets inhibited by CBX7, namely, E-cadherin (ECAD) and serine protease inhibitor Kazal-type 1 (SPINK1), were found increased in the bloodstream of patients with PH and were highly predictive of PH and clinically significant PH. Conclusions: We characterise the detrimental effects of increased hydrodynamic pressure on the sinusoidal endothelium, identify CBX7 as a pressure-sensitive transcription factor, and propose the combination of two of its reported products as biomarkers of PH. Impact and Implications: Increased pressure in the portal venous system that typically occurs during chronic liver disease (called portal hypertension) is one of the main drivers of related clinical complications, which are linked to a higher risk of death. In this study, we found that pathological pressure has a harmful effect on liver sinusoidal endothelial cells and identified CBX7 as a key protein involved in this process. CBX7 regulates the expression of E-cadherin and SPINK1, and consequently, measuring these proteins in the blood of patients with chronic liver disease allows the prediction of portal hypertension and clinically significant portal hypertension.

Cancer prognostics by direct detection of p53-antibodies on gold surfaces by impedance measurements.

The identification and measurement of biomarkers is critical to a broad range of methods that diagnose and monitor many diseases. Serum auto-antibodies are rapidly becoming interesting targets because of their biological and medical relevance. This paper describes a highly sensitive, label-free approach for the detection of p53-antibodies, a prognostic indicator in ovarian cancer as well as a biomarker in the early stages of other cancers. This approach uses impedance measurements on gold microelectrodes to measure antibody concentrations at the picomolar level in undiluted serum samples. The biosensor shows high selectivity as a result of the optimization of the epitopes responsible for the detection of p53-antibodies and was validated by several techniques including microcontact printing, self-assembled-monolayer desorption ionization (SAMDI) mass spectrometry, and adhesion pull-off force by atomic force microscopy (AFM). This transduction method will lead to fast and accurate diagnostic tools for the early detection of cancer and other diseases.

Vertically aligned carbon nanotubes for microelectrode arrays applications.

In this work a methodology to fabricate carbon nanotube based electrodes using plasma enhanced chemical vapour deposition has been explored and defined. The final integrated microelectrode based devices should present specific properties that make them suitable for microelectrode arrays applications. The methodology studied has been focused on the preparation of highly regular and dense vertically aligned carbon nanotube (VACNT) mat compatible with the standard lithography used for microelectrode arrays technology.

Direct Color Observation of Light-Driven Molecular Conformation-Induced Stress.

Although usually complex to handle, nanomechanical sensors are exceptional, label-free tools for monitoring molecular conformational changes, which makes them of paramount importance in understanding biomolecular interactions. Herein, a simple and inexpensive mechanical imaging approach based on low-stiffness cantilevers with structural coloration (mechanochromic cantilevers (MMC)) is demonstrated, able to monitor and quantify molecular conformational changes with similar sensitivity to the classical optical beam detection method of cantilever-based sensors (≈4.6 × 10-3  N m-1 ). This high sensitivity is achieved by using a white light and an RGB camera working in the reflection configuration. The sensor performance is demonstrated by monitoring the UV-light induced reversible conformational changes of azobenzene molecules coating. The trans-cis isomerization of the azobenzene molecules induces a deflection of the cantilevers modifying their diffracted color, which returns to the initial state by cis-trans relaxation. Interestingly, the mechanical imaging enables a simultaneous 2D mapping of the response thus enhancing the spatial resolution of the measurements. A tight correlation is found between the color output and the cantilever's deflection and curvature angle (sensitivities of 5 × 10-3  Hue µm-1 and 1.5 × 10-1  Hue (°)-1 ). These findings highlight the suitability of low-stiffness MMC as an enabling technology for monitoring molecular changes with unprecedented simplicity, high-throughput capability, and functionalities.