Organic Bioelectronic Devices for Metabolite Sensing.

Electrochemical detection of metabolites is essential for early diagnosis and continuous monitoring of a variety of health conditions. This review focuses on organic electronic material-based metabolite sensors and highlights their potential to tackle critical challenges associated with metabolite detection. We provide an overview of the distinct classes of organic electronic materials and biorecognition units used in metabolite sensors, explain the different detection strategies developed to date, and identify the advantages and drawbacks of each technology. We then benchmark state-of-the-art organic electronic metabolite sensors by categorizing them based on their application area (in vitro, body-interfaced, in vivo, and cell-interfaced). Finally, we share our perspective on using organic bioelectronic materials for metabolite sensing and address the current challenges for the devices and progress to come.

Polarization of disk electrodes in high-conductivity electrolyte solutions.

We investigate the polarization of disk electrodes immersed in an electrolyte solution and subjected to a small external AC voltage over a wide range of frequencies. A mathematical model is developed based on the Debye-Falkenhagen approximation to the coupled Poisson-Nernst-Planck equations. Analytical techniques are used for predicting the spatial distribution of the electric potential and the complex impedance of the system. Scales for impedance and frequency are identified, which lead to a self-similar behavior for a range of frequencies. Experiments are conducted with gold electrodes of sizes in the range 100-350 µm immersed in a high-conductivity KCl solution over five orders of magnitude in frequency. A collapse of data on impedance magnitude and phase angle onto universal curves is observed with scalings motivated by the mathematical model. A direct comparison with the approximate analytical formula for impedance is made without any fitting parameters, and a good agreement is found for the range of frequencies where the analytical model is valid.

Rapid and Sensitive Detection of Nanomolecules by an AC Electrothermal Flow Facilitated Impedance Immunosensor.

Conventional immunosensors typically rely on passive diffusion dominated transport of analytes for binding reaction and hence, it is limited by low sensitivity and long detection times. We report a simple and efficient impedance sensing method that can be utilized to overcome both sensitivity and diffusion limitations of immunosensors. This method incorporates the structural advantage of nanorod-covered interdigitated electrodes and the microstirring effect of AC electrothermal flow (ACET) with impedance spectroscopy. ACET flow induced by a biased AC electric field can rapidly convect the analyte onto nanorod structured electrodes within a few seconds and enriches the number of binding molecules because of the excessive effective surface area. We performed numerical simulations to investigate the effect of ACET flow on the biosensor performance. The results indicated that AC bias to the side electrodes could induce fast convective flow, which facilitates the transport of the target molecules to the binding region located in the middle as a floating electrode. The temperature rise due to the Joule heating effect was measured using a thermoreflectance imaging method to find the optimum device operation conditions. The change of impedance caused by the receptors-target molecules binding at the sample/electrode interface was experimentally measured and quantified in real-time using the impedance spectroscopy technique. We observed that the impedance sensing method exhibited extremely fast response compared with those under no bias conditions. The measured impedance change can reach saturation in a minute. Compared to the conventional incubation method, the ACET flow enhanced method is faster in its reaction time, and the detection limit can be reduced to 1 ng/mL. In this work, we demonstrate that this sensor technology is promising and reliable for rapid, sensitive, and real-time monitoring of biomolecules in biologically relevant media such as blood, urine, and saliva.

Self-Similar Response of Electrode Polarization for Binary Electrolytes in Parallel Plate Capacitor Systems.

Classical electrochemistry problem of polarization of an electrode immersed in a symmetric binary electrolyte and subjected to a small external ac voltage is revisited. The Nernst-Planck equations are simplified to the Debye-Falkenhagen equation, which is solved together with the Poisson equation, leading to analytical formulas for the space charge density and impedance of the system for two parallel plate electrodes. We then define a limit of thin electrical double layer and illustrate the emergence of the characteristic time scale, τc = λDL/D, a function of the Debye length, λD, the electrode separation distance, L, and the ionic diffusion coefficient D. Normalizing the impedance magnitude with the solution resistance and making the frequency dimensionless with the τc, we show that all analytical, numerical, and experimental data for different solution conductivities and electrode separation distances collapse onto a single curve. To account for the Stern layer effects, we conducted numerical simulations based on the modified Poisson-Nernst-Planck model and showed that the results agree with our analytical solution for a range of concentrations, with small discrepancies observed only above 0.1 M. Based on the proposed model, experimental impedance spectroscopy results at ac potentials can be used to obtain detailed knowledge of the corresponding surface (and space) charge densities on the electrodes.

Quantification of Cell Death Using an Impedance-Based Microfluidic Device.

Dielectric spectroscopy is a nondestructive method to characterize dielectric properties by measuring impedance data over a frequency spectrum. This method has been widely used for various applications such as counting, sizing, and monitoring biological cells and particles. Recently, utilization of this method has been suggested in various stages of the drug discovery process due to low sample consumption and fast analysis time. In this study, we used a previously developed microfluidic system to confine single PC-3 cells in microwells using dielectrophoretic forces and perform the impedance measurements. PC-3 cells are treated with 100 µM Enzalutamide drug, and their impedance response is recorded until the cells are totally dead as predicted with viability tests. Four different approaches are used to analyze the impedance spectrum. Equivalent circuit modeling is used to extract the cell electrical properties as a function of time. Principal component analysis (PCA) is used to quantify cellular response to drug as a function of time. Single frequency measurements are conducted to observe how the cells respond over time. Finally, opacity ratio is defined as an additional quantification method. This device is capable of quantitatively measuring drug effects on biological cells and detecting cell death. The results show that the proposed microfluidic system has the potential to be used in early stages of the drug discovery process.

Characterization of Temperature Rise in Alternating Current Electrothermal Flow Using Thermoreflectance Method.

Alternating current electrothermal flow (ACET) induced by Joule heating is utilized to transport biologically relevant liquids in microchannels using simple electrode designs. However, Joule heating may cause significant temperature rises, which can degrade biological species, and hence, ACET may become impractical for biomicrofluidic sensors and other possible applications. In this study, the temperature rise at the electrode/electrolyte interface during ACET flow is measured using a high-resolution, noninvasive, thermoreflectance imaging method, which is generally utilized in microelectronics thermal imaging applications. The experimental findings reveal that Joule heating could result in an excessive temperature rise, exceeding 50 °C at higher voltage levels (20 Vpp). The measured data are compared with the results of the enhanced ACET theoretical model, which predicts the temperature rise accurately, even at high levels of applied voltages. Overall, our study provides a temperature measurement technique that is used for the first time for electrode/electrolyte systems. The reported results are critical in designing biomicrofluidic systems with significant energy dissipation in conductive fluids.

Self-Similar Interfacial Impedance of Electrodes in High Conductivity Media: II. Disk Electrodes.

Electrode polarization effects were investigated using impedance spectroscopy measurements for planar and nanorod-structured gold disk electrodes at 100 Hz to 1 MHz frequency range and in 0.25 S/m to 1.5 S/m conductivity KCl solutions. Diameters of planar electrodes were varied from 50 µm to 2 mm to examine the effect of electrode size on impedance spectra. Normalizing the impedance magnitude with the spreading resistance and frequency with the characteristic time scale, all experimental data collapsed onto a universal curve, proving self-similarity. Experimental impedance results were compared well with that obtained from the numerical solution of Poisson-Nernst-Planck equations in axisymmetric domain. The influence of surface morphology was also investigated by generating cylindrical nanorods on a planar electrode. The 500 µm diameter electrode surface was covered with cylindrical nanorods with known height, diameter, and separation distance, which were characterized using scanning electron microscopy. The characteristic time scale for the nanorod-structured electrode increased by the surface enlargement factor obtained by cyclic voltammetry measurements. Self-similar interfacial impedance of electrodes was modeled using a constant phase element model. Current findings describe the coupled effects of electrode diameter, electrolyte conductivity, and electrode surface morphology on the impedance spectra of electrode/electrolyte system when the electric double layer between the nanorods does not overlap.

Atomic Scale Interfacial Transport at an Extended Evaporating Meniscus.

Recent developments in fabrication techniques have enabled the production of nano- and Ångström-scale conduits. While scientists are able to conduct experimental studies to demonstrate extreme evaporation rates from these capillaries, theoretical modeling of evaporation from a few nanometers or sub-nanometer meniscus interfaces, where the adsorbed film, the transition film, and the intrinsic region are intertwined, is absent in the literature. Using the computational setup constructed, we first identified the detailed profile of a nanoscale evaporating interface and then discovered the existence of lateral momentum transport within and associated net evaporation from adsorbed liquid layers, which are long believed to be at the equilibrium established between equal rates of evaporation and condensation. Contribution of evaporation from the adsorbed layer increases the effective evaporation area, reducing the excessively estimated evaporation flux values. This work takes the first step toward a comprehensive understanding of atomic/molecular scale interfacial transport at extended evaporating menisci. The modeling strategy used in this study opens an opportunity for computational experimentation of steady-state evaporation and condensation at liquid-vapor interfaces located in capillary nanoconduits.

Gold nanostructure microelectrode arrays for in vitro recording and stimulation from neuronal networks.

An ideal microelectrode array (MEA) design should include materials and structures which exhibit biocompatibility, low electrode polarization, low impedance/noise, and structural durability. Here, the fabrication of MEAs with indium tin oxide (ITO) electrodes deposited with self-similar gold nanostructures (GNS) is described. We show that fern leaf fractal-like GNS deposited on ITO electrodes are conducive for neural cell attachment and viability while reducing the interfacial impedance more than two orders of magnitude at low frequencies (100-1000 Hz) versus bare ITO. GNS MEAs, with low interfacial impedance, allowed the detection of extracellular action potentials with excellent signal-to-noise ratios (SNR, 20.26 ± 2.14). Additionally, the modified electrodes demonstrated electrochemical and mechanical stability over 29 d in vitro.

Electrical Impedance Measurements of Biological Cells in Response to External Stimuli.

Dielectric spectroscopy (DS) is a noninvasive technique for real-time measurements of the impedance spectra of biological cells. DS enables characterization of cellular dielectric properties such as membrane capacitance and cytoplasmic conductivity. We have developed a lab-on-a-chip device that uses an electro-activated microwells array for capturing, DS measurements, and unloading of biological cells. Impedance measurements were conducted at 0.2 V in the 10 kHz to 40 MHz range with 6 s time resolution. An equivalent circuit model was developed to extract the cell membrane capacitance and cell cytoplasmic conductivity from the impedance spectra. A human prostate cancer cell line, PC-3, was used to evaluate the device performance. Suspension of PC-3 cells in low conductivity buffers (LCB) enhanced their dielectrophoretic trapping and impedance response. We report the time course of the variations in dielectric properties of PC-3 cells suspended in LCB and their response to sudden pH change from a pH of 7.3 to a pH of 5.8. Importantly, we demonstrated that our device enabled real-time measurements of dielectric properties of live cancer cells and allowed the assessment of the cellular response to variations in buffer conductivity and pH. These data support further development of this device toward single cell measurements.

Self-Similar Interfacial Impedance of Electrodes in High Conductivity Media.

Electrode polarization (EP) happening due to accumulation of ions at the electrode/electrolyte interface is an inevitable phenomenon while measuring impedance spectrum in high conductivity buffers and at low RF spectrum. Well-characterized time scales elucidating the EP effect are important for the rational design of microfluidic devices and impedance sensors. In this Article, interfacial impedance at the electrode/electrolyte interface is investigated considering channel height and Debye length effects on characteristic time scale in a binary electrolyte solution using parallel plate electrode configuration. Experimental results reveal self-similarity of normalized electrical impedance as a function of the normalized frequency. The experimental results also match with numerical solutions obtained by finite element simulation of unsteady fully coupled Poisson-Nernst-Planck (PNP) equations. Furthermore, fractal shaped gold nanostructured electrodes are examined, and it has been proven that electric double layer (EDL) formed on porous electrode surfaces acts as a thick EDL and modifications to the characteristic time scale is necessary for porous electrodes. Finally, a constant phase element (CPE) model is proposed to account for the self-similar impedance spectrum, which can be used for different channel heights and solution conductivities.

Enhancement of dielectrophoresis using fractal gold nanostructured electrodes.

Dielectrophoretic motions of Saccharomyces cerevisiae (yeast) cells and colloidal gold are investigated using electrochemically modified electrodes exhibiting fractal topology. Electrodeposition of gold on electrodes generated repeated patterns with a fern-leaf type self-similarity. A particle tracking algorithm is used to extract dielectrophoretic particle velocities using fractal and planar electrodes in two different medium conductivities. The results show increased dielectrophoretic force when using fractal electrodes. Strong negative dielectrophoresis of yeast cells in high-conductivity media (1.5 S/m) is observed using fractal electrodes, while no significant motion is present using planar electrodes. Electrical impedance at the electrode/electrolyte interface is measured using impedance spectroscopy technique. Stronger electrode polarization (EP) effects are reported for planar electrodes. Decreased EP in fractal electrodes is considered as a reason for enhanced dielectrophoretic response.

Dielectrophoresis assisted loading and unloading of microwells for impedance spectroscopy.

Dielectric spectroscopy (DS) is a noninvasive, label-free, fast, and promising technique for measuring dielectric properties of biological cells in real time. We demonstrate a microchip that consists of electro-activated microwell arrays for positive dielectrophoresis assisted cell capture, DS measurements, and negative dielectrophoresis driven cell unloading; thus, providing a high-throughput cell analysis platform. To the best of our knowledge, this is the first microfluidic chip that combines electro-activated microwells and DS to analyze biological cells. Device performance is tested using Saccharomyces cerevisiae (yeast) cells. DEP response of yeast cells is determined by measuring their Clausius-Mossotti factor using biophysical models in parallel plate microelectrode geometry. This information is used to determine the excitation frequency to load and unload wells. Effect of yeast cells on the measured impedance spectrum was examined both experimentally and numerically. Good match between the numerical and experimental results establishes the potential use of the microchip device for extracting subcellular properties of biological cells in a rapid and nonexpensive manner.

Rough Gold Electrodes for Decreasing Impedance at the Electrolyte/Electrode Interface.

Electrode polarization at the electrolyte/electrode interface is often undesirable for bio-sensing applications, where charge accumulated over an electrode at constant potential causes large potential drop at the interface and low measurement sensitivity. In this study, novel rough electrodes were developed for decreasing electrical impedance at the interface. The electrodes were fabricated using electrochemical deposition of gold and sintering of gold nanoparticles. The performances of the gold electrodes were compared with platinum black electrodes. A constant phase element model was used to describe the interfacial impedance. Hundred folds of decrease in interfacial impedance were observed for fractal gold electrodes and platinum black. Biotoxicity, contact angle, and surface morphology of the electrodes were investigated. Relatively low toxicity and hydrophilic nature of the fractal and granulated gold electrodes make them suitable for bioimpedance and cell electromanipulation studies compared to platinum black electrodes which are both hydrophobic and toxic.

Eutectogels as a Semisolid Electrolyte for Organic Electrochemical Transistors.

Organic electrochemical transistors (OECTs) are signal transducers offering high amplification, which makes them particularly advantageous for detecting weak biological signals. While OECTs typically operate with aqueous electrolytes, those employing solid-like gels as the dielectric layer can be excellent candidates for constructing wearable electrophysiology probes. Despite their potential, the impact of the gel electrolyte type and composition on the operation of the OECT and the associated device design considerations for optimal performance with a chosen electrolyte have remained ambiguous. In this work, we investigate the influence of three types of gel electrolytes-hydrogels, eutectogels, and iongels, each with varying compositions on the performance of OECTs. Our findings highlight the superiority of the eutectogel electrolyte, which comprises poly(glycerol 1,3-diglycerolate diacrylate) as the polymer matrix and choline chloride in combination with 1,3-propanediol deep eutectic solvent as the ionic component. This eutectogel electrolyte outperforms hydrogel and iongel counterparts of equivalent dimensions, yielding the most favorable transient and steady-state performance for both p-type depletion and p-type/n-type enhancement mode transistors gated with silver/silver chloride (Ag/AgCl). Furthermore, the eutectogel-integrated enhancement mode OECTs exhibit exceptional operational stability, reflected in the absence of signal-to-noise ratio (SNR) variation in the simulated electrocardiogram (ECG) recordings conducted continuously over a period of 5 h, as well as daily measurements spanning 30 days. Eutectogel-based OECTs also exhibit higher ECG signal amplitudes and SNR than their counterparts, utilizing the commercially available hydrogel, which is the most common electrolyte for cutaneous electrodes. These findings underscore the potential of eutectogels as a semisolid electrolyte for OECTs, particularly in applications demanding robust and prolonged physiological signal monitoring.

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.

A single n-type semiconducting polymer-based photo-electrochemical transistor.

Conjugated polymer films, which can conduct both ionic and electronic charges, are central to building soft electronic sensors and actuators. Despite the possible interplay between light absorption and the mixed conductivity of these materials in aqueous biological media, no single polymer film has been utilized to create a solar-switchable organic bioelectronic circuit that relies on a fully reversible and redox reaction-free potentiometric photodetection and current modulation. Here we demonstrate that the absorption of light by an electron and cation-transporting polymer film reversibly modulates its electrochemical potential and conductivity in an aqueous electrolyte, which is harnessed to design an n-type photo-electrochemical transistor (n-OPECT). By controlling the intensity of light incident on the n-type polymeric gate electrode, we generate transistor output characteristics that mimic the modulation of the polymeric channel current achieved through gate voltage control. The micron-scale n-OPECT exhibits a high signal-to-noise ratio and an excellent sensitivity to low light intensities. We demonstrate three direct applications of the n-OPECT, i.e., a photoplethysmogram recorder, a light-controlled inverter circuit, and a light-gated artificial synapse, underscoring the suitability of this platform for a myriad of biomedical applications that involve light intensity changes.

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.

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.