Symposium SB06—Bioelectronic Materials and Devices for In Vitro Systems

Poly(ethylenedioxythiophene):poly(styrene sulfonate) (PEDOT:PSS) is the gold-standard active channel material for the organic electrochemical transistor (OECT). The PEDOT conjugated backbone features good redox stability in aqueous electrolyte, low oxidation potential and relatively small band gap energy [1]. However, PEDOT:PSS has two major drawbacks. The PEDOT backbone lacks biofunctionality, limiting its biocompatibility [2]. The dopant PSS is insulating and has been shown to leach out from the device over time, causing a significant drop in its electronic stability [3]. Fine tuning of the interface between the OECT device and the biological environment is crucial. Long-term electronic stability is a requirement for an electronic implant to efficiently operate at the interface. Therefore, there is a need for the design of functional materials to achieve tailored properties and long-term stability [4].
Here, we describe the design of graft and random copolymers based on functionalized EDOT monomers. Compared to PEDOT:PSS, we show that when used as active channel materials in OECTs, desirable properties are achieved including high volumetric capacitance, comparable mobility, and long-term stability with no decrease in drain current. Furthermore, we demonstrate post-functionalization of the film surfaces with biomolecules, enabled by the functional groups tethered on the PEDOT backbone. In conclusion, we show that careful design of the molecular structure of the conjugated polymer enables biofunctional and electronically stable active channel materials.
[1] L. Groenendaal, F. Jonas, D. Freitag, H. Pielartzik, J. Reynolds. Adv. Mater. 2000, 12, 481.
[2] D. Mantione, I. del Agua, A. Sanchez-Sanchez, D. Mecerreyes. Polymers, 2017, 9, 354.
[3] S-M. Kim, C-H. Kim, Y. Kim, N. Kim, W-J. Lee, E-H. Lee, D. Kim, S. Park, K. Lee, J. Rivnay, M-H. Yoon. Nat. Commun. 2018, 9, 3858.
[4] K. Fidanovski, D. Mawad. Adv. Health. Mater. 2019, 8, 1900053; C. Baker, K. Wagner, P. Wagner, D.L. Officer, D. Mawad. Adv. Phys-X. 2021, 6, 1899850.

Poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS) has been considered as one of the promising soft materials for emerging bioelectronic devices. There is increasing interest in the use of drop-on-demand inkjet printing as an additive manufacturing process. This talk presents the rheological properties, inkjet processes and bioelectronic applications of the polymer-containing printable ink. We show that the aqueous solution of PEDOT:PSS is strongly shear-thinning, and discuss how it can produce satellite-free inkjets. Furthermore, we characterize the rheological properties of a crosslinker (3-glycidyloxypropyl) trimethoxysilane-containing PEDOT:PSS at high frequencies, and investigate how the interactions of the polymers in the solution even at room temperature affect printability. Based on the optimized ink formulation and printing process, we demonstrate a technique to inkjet-print thin-film resistors of PEDOT:PSS on plastic which enables us to achieve five orders of magnitude in resistance value with high repeatability, while the feature size of the resistors remains unchanged. Finally, we present inkjet-printed organic voltage amplifiers by integrating active and passive electrical components on a single, highly flexible substrate for in vivo brain activity recording. Drop-on-demand printing facilitates fine-tuning of the voltage amplification properties while monolithic integration of the printed circuit achieves significant noise reduction. Finally, we validate the conformable brain-integrated organic voltage amplifiers as electrocorticography devices in a rat in vivo model, showing their ability to record local field potentials in anaesthetized animals.

We examine the neuromorphic behavior of organic electrochemical transistors (OECTs) based
on polymer blends of a mixed conductor (PEDOT:PSS) and an ion conductor (PSSNa). We
show that the addition of an ionic conductor into PEDOT:PSS decreases the information
retention time in paired-pulse depression (PPD) experiments. Detailed studies of transient
properties demonstrate that the relevant time scales for the neuromorphic response are
determined by an ionic response of the device. These results bring to the forefront OECTs as
devices with neuromorphic properties that are readily engineered.

Organic electrochemical transistors (OECTs) have shown great potentials for biosensing and bioelectronics because of their low working voltage and compatibility with an aqueous solution. The operation of OECTs is based on modulating the drain current by injecting/extracting ions into/from the active channel by gate voltage, which leads to doping and de-doping of it. The repetitive OECT measurements often lead a performance degradation, which is not favored for biosensing applications. In this work, we show that the magnitude of gate voltage in the off state (V_{g, off}) is crucial to the stability of the transfer curve of an OECT based on interdigitated electrodes (IDEs). A high V_{g, off} such as +0.5 V promotes a larger scale of Na⁺ ion injection, leading to a recognizable drain current decay in the transfer characteristics of the PEDOT:PSS OECTs under repeated operations. By lowering the V_{g, off} to +0.2 V, the OECTs can be stabilized at the cost of a lower on/off ratio. The crosslinking of PEDOT:PSS by GOPS can reduce the drain current decay and improve stability. For instance, crosslinked OECTs show a stable on/off ratio under a very demanding device operation voltage condition (V_g,off = +0.5 V, V_ds = -0.5 V). Control experiments exclude the possibility of PEDOT:PSS film decomposition as the reason for drain current decay. So the repetitive Na⁺ ion intercalation at high V_{g, off} is the most probable cause of the decay. The improved stability is most likely due to the suppression of the microstructural damages induced by expansion-shrinking during the switching between the “doped” and “de-doped” states.

Despite the non-conventional applications beyond thin film devices, there exist very few methods for constructing 3-dimensional structures based on conjugated polymers without sacrificing their intrinsic electrical/electrochemical properties. Herein, we report on a scalable method for fabricating highly conductive/capacitive 3-D architectures using pure poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS) microstructures without additives. First, crystalline PEDOT:PSS microfibers were fabricated via a conventional wet-spinning process and cut into smaller segments by homogenization in water. These highly-conductive/capacitive building blocks were self-fused to form monolithic mesh structures reversibly or irreversibly by inducing water swelling, chain entanglement, and crystallization in a controlled manner. The resultant porous mesh structures showed good mechanical flexibility and excellent electrical/electrochemical characteristics which could be modulated by controlling the loading of constituent microfiber segments. Finally, we demonstrated the fabrication of free-standing 3-D microfiber mesh structures with arbitrary shapes and their application to bioelectrical signal recording on live tissues without adhesives.

Organic electrochemical transistor (OECT) has excellent transconductivity compared to field-effect transistor. It also has good operation compatibility in aqeuoust environemt including implanted bioconditions. In organic electrochemical transistor (OECT), the ion of electrolyte penetrates into the organic channel and changes the conductivity. Research using the OECT sensor for sensing bio electric signals and bio materials is being actively conducted. However, OECT has a slower response time and a shorter lifespan than conventional transistors.
Here, we mixed PEDOT:PSS with polyacrylamide and electropin to produce nanofiber. This mixture have structural advantages over the existing film channel-type OECT by using nanofiber as a channel. First, it has a larger surface area in the same dimension than in the existing film type channel. The increase in the surface area leads to the increase in the penetration area of the ion, making it easier for the ion to penetrate. This allows for a faster response time(transient time) in terms of ion sensing. Second, it is stable in an underwater environment(in vitro) using polyacrylamide. It was confirmed that there was little change in performance even after 36 months in DI water. At room temperature in the phosphate buffer solution solution, the change in conductivity was maintained at around 10% even after 2 months. In addition, it was confirmed that there was little change in conductivity in an experiment in which 5000 cycles of repeated pulse signals were loadded. Third, PEDOT:PSS/polyacrylamide nanofiber is stretchable. Swelling and contraction occur in the process of dimethyl sulfoxide(DMSO) secondary doping in polyacrylamide. The crystallization of PEDOT:PSS occurs and its conductivity increases through DMSO secondary doping. In the case of PEDOT:PSS/polyacrylamide nanofiber, stretchability and conductivity are improved. In addition, there is no change in resistance to tensile stress, so it can be used as a stretchable sensor

Noble metals are well known for their high electrical conductivity, chemical stability and lack of native oxide layers. In bioelectronics, they are used in interfaces for recording and stimulation in cardiac pace makers, deep brain stimulators, and peripheral nerve interfaces. High effective surface area and low impedance are critical parameters in these applications. Porosification or roughening are often employed strategies to yield high interfacial area without increasing of geometric area. Most frequently they are based on electrochemical etching/deposition, or orthogonal dissolution of a codeposited metal/polymer template, yielding porous structures. These procedures are problematic, as they require multiple steps, materials, organic solvents or electrochemical treatments. In our work we developed a method for fabrication of high-surface area noble metal coatings based on reactive sputtering of the respective metal with mixture of Ar:O₂. Although this is a well-established approach for deposition of oxides layers such as Al₂O₃ or WO₃, its use for oxides of noble metals is rare. We show that for Au, Pd or Pt, it yields a (meta)stable oxide which, after chemical reduction, produces a conducting surface of high electrochemical capacitance and low impedance. We compare different reduction conditions and electrochemical parameters between the three noble metals. We show remarkable simplicity and cleanliness of our approach, which require a minimal number of steps, utilizes mild reduction conditions and does not introduce particulate impurities. Thanks to compatibility with wafer-scale microfabrication processes, we believe that our method can be particularly promising for microelectrodes used in bioelectronics applications.

Engineering neuronal circuits on artificial substrates using external parameters provides a novel way to understand the mechanisms underlying circuit formation in the brain [1]. It also provides insights into designing regenerative implants to interface with the nervous system. In this presentation, I will demonstrate the use of semiconducting nanowires as topographical cues to guide the formation of functional neural networks. Here I will present vertically aligned semiconductor nanowires for guiding growth of neural networks in neuronal cell cultures. Our results show that nanowires act as nanoscale topographical cues for neuronal growth, resulting in a directional growth of the processes and highly interconnected neuronal network. Our studies confirm that the alignment of cellular processes along nanowire patterns produces a highly interconnected neural network and correlates with a synchronized activity between neurons [2]. I will also present some of the recent insights into the mechanisms behind these observations where we have explored the effect of electronic properties of the substrates on the growth and function of neural networks.

[1] V Raj et al, Understanding, engineering, and modulating the growth of neural networks: An interdisciplinary approach Biophys Rev (2021)
[2] V. Gautam, et al, Engineering highly interconnected neuronal networks on nanopillar arrays Nano Lett. (2017).

Three-dimensional (3D) cell culture based in vitro models shown great promise and able to replicate partially the development and complexity of in vivo nerve tissue^1,2,3. The functional characterization of 3D cell cultures is challenging. Because, most of the in vitro neural interfacing technologies, e.g. 2D microelectrodes arrays (MEAs) are developed for 2D cell cultures, where it limits the access to the networks within the cultures⁴. Hence, high-performance electrophysiological platforms that allow seamless integration with soft and stable tissue system, and multi-directional bio interfacing capabilities for long term are required.
In this context, we report for the first time the fabrication of ‘Stargate’ look alike flexible and hollow micro-ring electrode array (SG MEA) with hole at the centre (40-µm to 150-µm diameter). The SG MEA was microfabricated on a 20-µm thick layer of flexible and biocompatible parylene C configured with 4 to 6 microelectrode sites. All gold electrode rings were coated with the conducting polymer poly(3,4-ethylenedioxythio-phene):poly(styrene-sulfonate) (PEDOT:PSS) to lower the impedance (5-8 kΩ) and obtain a better signal-to-noise ratio during extracellular recordings. The hollow ring shape of SG electrode architectures, with geometrical surface area of 289 µm² to 1299 µm² were designed specifically in such a way that it offers more freedom to the internal dynamics of cell cultures and multidirectional sensing of neuron activities within the 3D neuronal network, from both front and back-end of the SG MEA.
To monitor the extracellular recordings of spontaneous and evoked 3D neuronal activities from within, the SG MEA was incorporated vertically to the cell culture chamber. The neuronal cultures made up of rat embryonic hippocampal cells (E18) were grown in 3D, guided with porous silica beads assembly⁵. Silica beads (40-µm diameter) provide a growth surface to move, transfect and culture highly differentiated neurons without disrupting their membrane integrity. Consequently, it offers distinct subsets of neurons having constrained connectivity on different beads and consistent mechanical properties to keep them intact for longer periods. Later, the long term, stable and comprehensive 3D neuronal network recording was demonstrated over the course of neuron culture development at single cell resolution. The obtained results shows that our SG MEA combined with 3D monitoring approach holds a great potential in understanding the neuronal circuit formations and dynamics in multiple dimensions.
References
Bissell et al. Organoids: A historical perspective of thinking in three dimensions. J. Cell Biol. 216 (2016), 31–40
Pasca, S. The rise of three-dimensional human brain cultures. Nature 553 (2018), 437–445
Vallejo-Giraldo, Catalina et al. Hydrogels for 3D Neural Tissue Models: Understanding Cell-Material Interactions at a Molecular Level. Front Bioeng Biotechnol, 8 (2020), 601704.
K Tasnim et al. Emerging Bioelectronics for Brain Organoid Electrophysiology, J. Mol. Biol. (2021), 167165, ISSN 0022-2836
Pautot et al. Colloid-guided assembly of oriented 3D neuronal networks. Nat. Methods 5 (2008), 735–740.

Circulating tumor cells (CTCs) are cancer cells which reside in the bloodstream, often entering the circulatory system after detaching from tumors. CTCs promote cancer proliferation and metastasis. They display characteristics of primary tumor cells, including surface protein expression. Programmed death ligand 1 (PD-L1) is often upregulated on CTC surfaces, making it an attractive target for immunoaffinity-based capture. PD-L1 helps prevent immune cell response by reducing proliferation of antigen-specific T-cells and apoptosis in regulatory T-cells, largely allowing CTCs to evade immune response. CTCs are rare events in blood, ~10 CTCs/10⁹ blood cells. Anti-PD-L1-coated magnetoelastic (ME) sensors are being investigated for their ability to capture and detect CTCs. ME sensors are made from a magnetostrictive material, a ferrous alloy formed into an amorphous ribbon structure which responds based on the Joule effect: it changes in dimension in response to a magnetic field. Under an alternating magnetic field, the ME sensors stretch and compress, allowing establishment of a resonance frequency. This frequency is inversely related to the mass loading on the sensor surface. This effect allows for differentiation of the signals between bare and mass-loaded sensors, providing simple wireless CTC detection. This study investigates the efficacy of ME sensors in specific, sensitive CTC capture, necessary for early diagnosis, cancer phenotyping, and personalized cancer treatments.

Redox reactions in proteins, mediated by the electron transfer, are the engine that drives crucial life-sustaining functions including respiration, photosynthesis, metabolism, and cell apoptosis. Thus, the ability to modulate the redox proteins through an external stimulus presents a unique opportunity in attuning the system. Piezoelectric nanoparticles offer underexplored advantage of the electrical interaction under external acoustic waves; ultrasound offers biosafety and long penetration depth. In this work, we present the acousto-nanodevice capable of modulating the redox state of a redox protein (i.e., reduced cyt c) under ultrasonic irradiation. To enable the unidirectional electron transfer under alternating fields, we designed the device to have asymmetric surfaces. Our results clearly demonstrated that protein oxidation occurs only when asymmetrically functionalized acousto-nanodevice was activated by ultrasound. We also experimentally confirmed that the acousto-nanodevice may engineered to be either an oxidation or reduction agent, depends on the types of the surface metal. Based on the various control experiments, we postulate the working mechanism of the acousto-nanodevice is that the alternating electrical polarization of BTO nanoparticles can tentatively modulate the workfunction of the metal, thereby inducing unidirectional electron transfer from the protein redox centers to the acousto-nanodevice.

As the applications of microfluidics expand into more diverse fields, the fluid channels are now being interfaced/incorporated with methods/devices beyond the traditional pumping devices. Ultrasonic, thermal, electric, and magnetic flow control devices have been interfaced with or incorporated into microfluidic systems to achieve new functionalities such as particle/cell manipulations, drug delivery, and molecular diagnostics. Among those, embedded plasmonic metal nanoparticles, especially exploiting the self-formation through in-situ reduction of metal ions by crosslinking reagents, within the polydimethylsiloxane (PDMS) fluidic channel have been exploited for optical sensing applications and photothermal heating. Typical fabrication methods for these methods are liquid phase deposition of metal ion solution into uncured PDMS or submerging cured PDMS slabs in metal ion solution for a prolonged time. While effective, these methods offer little capability of patterning, ultimately limiting the applicability. In this work, we present monolithic inkjet-printed plasmonic structures for microfluidics, where the plasmonic photothermal elements are directly printed on to a PDMS microfluidics channels, fabricated by injection molding. Different carrier solutions for the metal ions were experimentally explored for the optimal printing conditions; we found that the printing condition depends on the surface tension and dipole moments of the solvent, which determine the mixing enthalpy and entropy among metal ions, solvent, and un-crosslinked PDMS. We demonstrated the monolithic inkjet printing of the plasmonic structure using the optimized printing conditions and injection molding, realizing different structures with various functions such as pumping, thermal lysing of viruses, mixing, and polymerase chain recombination.

Engineered neuronal networks have become a valuable tool to study neural cultures in vitro. The ability to control the growth and connectivity of neuronal networks in a regulated environment allows to study basic processes such as memory formation and learning, or to test the effect of drugs on the nervous system. However, most engineered neuronal networks are cultured on stiff substrates, not reflecting the soft and stretchable nature of the majority of biological tissues.
To address this issue, we present a modular device interfacing stretchable electronics and polydimethylsiloxane (PDMS) microstructures to study the mechanobiology of engineered neuronal networks in vitro. The device fabrication is based on two previously published technologies developed in our group: firstly, a stretchable multielectrode array (MEA) is produced by the patterning of porous polyvinylidene difluoride (PVDF) membranes with photolithography, followed by filtration of nanowires and nanoparticles through the patterned areas to create the tracks and electrodes. These conductive patterns are then transferred on semi-cured PDMS in a layered fashion to form the stretchable MEA¹. The electrodes are characterized using cyclic voltammetry and impedance spectroscopy. Then, a PDMS axon-guiding microstructure² is aligned on the multielectrode array and glued onto it using a thin film of diluted PDMS. The axon-guiding microstructure is produced using a standard soft lithography process: two-layer SU-8 masters are fabricated by spin coating and patterning. PDMS is spin-coated on the masters, resulting in an approximately 250 µm thick patterned microstructure. The first, top layer of the structure has circular landing spots open on top to enable the placement of neurons and neural spheroids into the desired nodes of a network. The second, bottom thin layer is patterned so that axons from neurons seeded in different nodes gradually merge with each other unidirectionally to form an artificial nerve, while cell bodies cannot penetrate into this six µm thick closed compartment of the microstructure. The mechanical properties of the device are investigated by performing a peeling test to characterize the interface bonding strength between the two layers. In addition, a cyclic stretching test is carried out to characterize the device electromechanically.
Neurons can then be cultured in the microstructure and their axons grow in a controlled and confined manner in microchannels, forming engineered neuronal cultures. The biocompatibility of the device is tested by live/dead staining. We envision using the stretchable MEA to stimulate the neurons electrically under various strain conditions for mechano-neurobiology studies.
References:
1. Renz, A., Lee, J., Tybrandt, K., Brzezinski, M., Lorenzo, D., & Cerra Cheraka, M. et al. (2020). Opto-E-Dura: A Soft, Stretchable ECoG Array for Multimodal, Multiscale Neuroscience. Advanced Healthcare Materials, 9(17), 2000814
2. Forró, C., Thompson-Steckel, G., Weaver, S., Weydert, S., Ihle, S., & Dermutz, H. et al. (2018). Modular microstructure design to build neuronal networks of defined functional connectivity. Biosensors And Bioelectronics, 122, 75-87

Delivering biomolecules into living cells has become an important challenge in medical and biological fields. Conventional techniques such as virus vector and electroporation are utilized commonly for small molecule delivery, but still remain several problems of low efficiency and low viability for large molecules such as biological proteins and organelles. To overcome this problem, we demonstrate the macromolecular delivery into adhesive cells with metallic nanotube ducts. The metallic nanotubes were developed by an electroless plating of gold onto track-etched polycarbonate template and wet/dry etching [1]. To insert Au nanotubes into the cells (such as HeLa and NIH3T3), we developed the height control system with the fluorescent microscope.
Furthermore, by coating the surface of this nanotube with a conductive polymer (PEDOT), we developed a novel Au nanotube/PEDOT, which is hybrid nanotubes (HyNTs), that promotes the flow of ions at an applied voltage (about 50 mV) below the cell membrane potential [2]. As a result, we have succeeded in developing an electric nanoinjector that delivers macromolecules such as green fluorescent protein (GFP) and DNA plasmid into cells.

[1] Bowen Zhang, Yiming Shi, Daisuke Miyamoto, Koji Nakazawa, Takeo Miyake, “Nanostraw membrane stamping for direct delivery of molecules into adhesive cells”, Scientific Reports, 9, 6806, 2019.
[2] Bowen Zhang, Dinuo Zheng, Shi Yiming, Kazuhiro Oyama, Masahiro Ito, Masaomi Ikari, Takanori Kigawa, Tsutomu Mikawa, Takeo Miyake, “High-Efficient and Dosage-Controllable Intracellular Cargo Delivery through Electrochemical Metal-Organic Hybrid Nanogates”, Small Science, 10, 1002, 2021.

To overcome the problems of stem cell therapies, direct reprogramming which means reprogramming the somatic cell into a specific cell directly without passing through another cell stage is a new approach. However, functional maturation still remains challenging. Therefore, the aim in this study is to apply electrical stimulation to promote direct reprogramming with maintaining the three-dimensional spheroid culture of chemically induced cardiomyocytes. To do that, a novel electrode platform was designed with the dimensions in consideration of stability of both micropillar and spheroid. The average size of spheroids is around 230~260 µm, therefore, the diagonal distance between two micropillars was designed at 183.85 µm and the height of the micropillars was designed at 80 µm in order to avoid spheroid attachment on the bottom of the device. The micropillars were fabricated with deep reactive-ion etching (DRIE) and confirmed with scanning electron microscope (SEM). Through computer simulation technology (CST) Studio Suite, the electrical field of micropillar electrode and film electrode was simulated and the electrical stimulation protocol was optimized (biphasic 1V, 10Hz every 10 seconds for 1 hour). The improvement of the electrophysiological properties of reprogrammed spheroids through the highly efficient electrical stimulation by a micropillar electrode arrays was demonstrated through the comparison with the group without the electrical stimulation.

The recent development of bio-integrated technology based on wearable electronic devices enables continuous access to real-time physiological and electrophysiological data wirelessly. Electrophysiology, which was mainly used in restricted medical environments, is now actively used for human-machine interaction or continuous health monitoring in unrestricted daily life.
Lower skin-electrode impedance is the main key factor for the successful measurement of bioelectrical signals. The factor that most affects the impedance is the stratum corneum (SC), which is the outer layer of the epidermis composed of dead cells. Dead cells have poor conductivity compared to living cells and increase the impedance extremely. Also, non-conformal contact between skin and electrode due to irregular skin surface causes high impedance as well. Therefore, it is possible to reduce the impedance by increasing the unit contact are between the electrode and the surface of the curved skin. Wet electrodes normally utilized for medical purposes can reduce impedance by using electrolyte gel to form an electrical bridge and cover the curved human skin. However, the wet electrode is inappropriate for long-term measurements because the duration of the effective impedance is until the gel dries. For this reason, the development of dry electrodes based on micro-electro-mechanical systems that do not use electrolyte gel and do not require skin preparation have been actively developed. There are two types of dry electrodes that are being actively studied recently: epidermal and needle electrodes.
Epidermal electrodes can conformally contact the curved human skin and increase contact area due to its "skin-like" properties such as lightweight, ultrathin, low-modulus, and stretchable. It is advantageous for long-term measurements and less motion artifact. Although the epidermal electrode is in very close contact with the epidermis, it has a limitation in that it only contacts the stratum corneum of the epidermis which acts insulator for the electrical signals. In addition, since it is in contact with the skin only by the van der Waals force, there is a limit in the adhesive force. While the epidermal electrode focuses on the contact area to reduce the impedance, the microneedle electrode focused on overcoming the limitations of the stratum corneum. Since the needle penetrates the stratum corneum, the effect of the stratum corneum on impedance can be removed. However, volunteers feel pain because the needle continues to penetrate the skin, and it is difficult to last long. Also, since the substrate holding the needle is required, it has the disadvantage of being thicker and heavier than the epidermal electrode.
In this paper, we present a tattoo-like, epidermal microneedle electrode with different electrode mounting approaches. This new electrode penetrates the stratum corneum, dramatically lowering the impedance, yet painless to the user. The flat part of the electrode offers solid adhesion between electrode and skin that helps the long-term measurement and lower motion artifact. A water-soluble PVA serves as a mold and temporary support. Rigid PVA can penetrates the stratum corneum and implants a very thin epidermal electrode into the epidermis, and then dissolved by water. As a result, a very thin Parylene/Cr/Au electrode alone penetrates the stratum corneum. An impedance test was performed by attaching the electrode to the skin, and it was confirmed that this value had a sufficiently low impedance. In addition, by measuring EEG, ECG, EMG in a long term in the environment of daily life, the feasibility of the application of the electrode was proved.

Globally, cancer remains the second leading cause of death and millions of people still suffer from it, and a third of them die every year. Developing new anticancer agents and determining their activities are an essential part of drug discovery, but it takes a long time and needs abundant resources, resulting in the increasing demand for highly effective techniques. To examine the anticancer efficacy of novel drugs, cell staining/counting methods such as Cell Counting Kit-8 (CCK-8), thiazolyl blue tetrazolium bromide (MTT) assay, and trypan blue exclusion have been commonly carried out to detect cell viability. However, those assays have a limitation in assessing the efficiency of colored drugs due to a possibility of signal interference at the specific wavelength. It also requires time-consuming and complicated sampling processes and invasive chemical reagents for measurements of cellular metabolic activities. For these reasons, alternative sensing techniques using optics, electrochemistry, and quartz crystal microbalances have emerged for in vitro toxicological evaluation. Among them, the electrochemical strategy, which features high sensitivity, fast response time, easy miniaturization, real-time monitoring, and cost-effectiveness has been exploited to analyze the anticancer activities with the reduced time and cost in the early stage of drug development. In particular, an electrochemical cell-based biosensor, called a cytosensor, has been regarded as a fast, sensitive, non-invasive, and label-free technique to monitor the various cell types such as pluripotent stem cells and cancer cells. Based on the oxidation/reduction reaction of several biomolecules (e.g., intracellular redox proteins and metalloproteins), distinctive electrochemical redox signals from each cell type were found to be generated and recorded at specific potentials, and these can be applied to investigate the cancer cells’ response to the anticancer drugs.
In this study, we present an electrochemical cytosensor that enables rapid and sensitive assessment of the effects of an anticancer drug, saponin, on the most aggressive form of skin cancer, called human malignant melanoma (SK-MEL28). Electrochemical deposition of gold nanoparticles (AuNPs) onto fluorine-doped tin oxide (FTO) electrodes was conducted to promote their electrocatalytic properties. The AuNPs/FTO platforms were further decorated with biocompatible poly-L-lysine (PLL) to facilitate cell adhesion and growth. By combining the nano and biocompatible materials, direct electron transfer between immobilized cancer cells and the fabricated electrode occurs and both selectivity and sensitivity of the cytosensor were improved. The electrical signal of SK-MEL28 cells was obtained from cyclic voltammetry and differential pulse voltammetry and showed clear linearity (R² = 0.9952) relative to the number of cells, suggesting that the intensity of the cathodic peak current of the cytosensor can be used to detect the number of SK-MEL28 cells. Moreover, the anticancer efficacy of saponin on SK-MEL28 cancer cells was clearly proved to be effective at concentrations higher than 20 μM, which was highly in agreement with the conventional assays. The developed electrochemical cytosensor for assessing anticancer effects of drugs enabled sensitive (Limit of quantification: 2,880 cells/device), rapid (< 2 min), and non-invasive measurements, thus providing a simple and efficient way to do research on the evaluation of novel drug candidates and encourage the development of powerful anticancer reagents.

Bioelectronic devices that can interface electronics with biological system can be used as actuators to control biological processes. In this work, we present a bioelectronic platform that can control pH and [K+] in buffered media solutions and implement a fluorescence sensing based feedback control algorithm in order to close the loop between sensing and actuation. We also report systematic investigation of various aspects of device performance using electrical characterization techniques and computational modeling. This work can be used in a wide range of novel therapeutic applications. We intend to extend this research further by performing actuation on cells and controlling their response using closed-loop control. This can be further expanded to create systems that enable cell differentiation and accelerated wound healing

Transistor-based signal amplification technologies have been extensively applied to develop a variety of sensors to detect optical, electrical, chemical, and even biological signals with an exceptionally high sensitivity in real time. However, without adequate signal filtration, the selectivity of the resulting sensors is inevitably poor; in particular, as bioanalytes are in a complex mixture, it would be significantly challenging to selectively detect the desired ions, metabolites, or proteins in real time with no sample purification process. In achieving target-selective signal amplification, size-dependent physical filters, including metal-organic frameworks and porous graphenes, have been successfully combined with transistor-based biosensors to date [1], but the size filter-embedded biosensors still suffer from distinguishing different molecules of similar size, which are very common in a biological sample. To address this issue, we aimed to selectively regulate the transport of target molecules relying on molecular recognition rather than sieving separation, and in this work, by mimicking ion channels in biological membranes, we newly developed K⁺ ion-selective membrane filter and integrated it with our hydrogel ionic transistor. Briefly, by chemically modifying G-quadruplex nucleic acid nanostructures that contain K⁺ binding motifs, we created artificial K⁺ channels, which were subsequently inserted in lipid bilayers. Due to the lateral hydrophobic modification of the channel, its entry and exit points were hydrophilic only, allowing ionic flows perpendicular to the lipid membranes. As a result, while anion transport was perfectly blocked (~100%) through our affinity-based membrane filter, K⁺-preferred ion transport among similar-sized alkali metal ions was successfully observed. As the K⁺-selective membrane filter was readily integrated with the hydrogel ionic transistor that enables ion-to-ion amplification, we succeeded in monitoring K⁺-specific ion signals during cellular growth in real time.

References:
[1] Koenig, Steven P., et al. "Selective molecular sieving through porous graphene." Nature nanotechnology 7.11 (2012): 728-732.

A novel device construction and experimental setup is proposed, facilitating in-situ fluid and cellular analysis by microfluidic impedance spectroscopy. The device structure utilises advanced additive manufacturing techniques such as aerosol jet printing, offering the opportunity to tailor devices to specific applications whilst maintaining low production costs [1]. The device consists of a pair of silver interdigitated electrodes coated in a protective polyimide layer, aerosol jet printed onto a glass substrate. Microfluidic channels produced by stereolithography enable analysis and characterisation of small sample volumes.

Impedance spectroscopy alleviates the need for optical, electrochemical or other detection and diagnostic techniques, instead probing the sample more directly [2]. Thus, through the on-chip integration of electronics and biological components, the device structure can be truly ‘lab-on-chip’. Despite this significant advantage, the use of impedance spectroscopy to characterise liquids is currently somewhat less widespread than its use for the analysis of solids, where its effectiveness has been proven.

In addition to maintaining laminar flow, the nature of a microfluidic system lends itself well to temperature-controlled experiments. The small length scales enable temperature to be accurately controlled and precisely measured. The significance of this extends beyond the analysis of temperature-sensitive biological samples; impedance spectra acquired at a range of temperatures can provide additional information about samples, and the interactions and mechanisms within them.

Previous studies have demonstrated a strong link between the concentration of important biological analytes and the dielectric constant of the a fluid [3,4]. Thus, the system could characterise complex fluids to determine the concentration of specific target analytes, and monitor changes over time. In addition, it could be utilised to investigate cellular metabolic activity, via measurements of the dielectric properties of the surrounding media. For example, this could facilitate the development of new drugs, or investigations of drug resistance in particular cell types.
References:
[1] N. Catic et al., App. Mat. Tod, 19 (2020), p.100618
[2] J. MacDonald, J. Electroanal. Chem, 223 (1987), p.25
[3] A. Dutta et al., Sens. Trans., 1 (2020), p.11
[4] L. Lima et al., J. Molec. Liq., 241 (2017), p.530

Graphene has triggered tremendous interest in biosensing due to its superior electrical, mechanical and optical properties. For example, the high electron mobility and transparent nature of graphene allow for sensing of electrical activities with simultaneous high-resolution optical imaging. Here, we report on real-time extracellular electrical activity recording of retinal ganglion cells (RGCs) with flexible graphene electrodes integrated into perforated microfluidic platforms, which represents the first attempt of applying flexible graphene probes in retina study that can simultaneously monitor extracellular spiking activities from multiple channels with a high temporal and spatial resolution. Our results show under identical conditions, the graphene electrodes exhibit a higher electrical sensitivity than the gold counterparts with approximately 2.5 fold enhancement in the recorded spiking amplitude. Moreover, when RGCs are stimulated with high K⁺ concentration, they display a strong response with the firing rate first increasing and then ceasing, likely be due to the potassium-induced neural depolarization. Under light stimulation, we observed three distinct response patterns: ON, OFF, and ON-OFF, which reflects how different types of RGCs behave in response to light mediated by cone-shaped photoreceptors. Moreover, the observed spiking waveforms can be divided into two groups: the biphasic waveform usually as a result of direct contact between the electrode and soma, and the triphasic waveform that is likely from an isolated axon in contact with the electrode. Our results suggest that graphene electrodes can be a promising candidate for the electrophysiology studies of the retina and offer a route to engineering two-dimensional materials based flexible biosensors.

Light-based therapy such as photobiomodulation (PBM) reportedly produces beneficial physiological effects in cells and tissues. However, most reports have focused on the immediate and instant effects of light. Considering the physiological effects of natural light exposure in living organisms, the latent reaction period after irradiation should be deliberated. In contrast to previous reports, we examined the latent reaction period after light exposure with optimized irradiating parameters and validated novel therapeutic molecular mechanisms for the first time. we demonstrated an organic light-emitting diode (OLED)-based PBM (OPBM) strategy that enhances the angiogenic efficacy of human adipose-derived stem cells (hADSCs) via direct irradiation with red OLEDs of optimized wavelength, voltage, current, luminance, and duration, and investigated the underlying molecular mechanisms. Our results revealed that the angiogenic paracrine effect, viability, and adhesion of hADSCs were significantly intensified by our OPBM strategy. Following OPBM treatment, significant changes were observed in HIF-1α expression, intracellular reactive oxygen species levels, activation of the receptor tyrosine kinase, and glycolytic pathways in hADSCs. In addition, transplantation of OLED-irradiated hADSCs resulted in significantly enhanced limb salvage ratio in a mouse model of hindlimb ischemia. Our OPBM might serve as a new paradigm for stem cell culture systems to develop cell-based therapies in the future.

Brain organoids have emerged as a substitution of the human brain for clarifying the origins of neurodevelopmental diseases, by understanding their electrophysiological developments and mechanisms in three-dimensional (3D), volumetric neural networks. However, existing devices to record neural signals either contact the surface of organoids or involve the section or invasion by hard materials, which limit the chronic analysis of soft brain organoids.
Herein, we present a liquid 3D microelectrode array (MEA) by high-resolution 3D printing of biocompatible metals for chronic electrophysiological analysis of brain organoids. Recording neural signals from the organoid with this printed device is non-destructive and non-invasive owing to the soft mechanical properties of liquidous metallic probes, which makes long-term analysis possible. The height of these liquid 3D probes is controllable to monitor electrophysiological signals from entire 3D spatial points in the organoid. Furthermore, magnetic control of these liquid 3D probes facilitates the change in their tip position, which can enhance the resolution of spatial points where these bioelectrodes are located inside the organoid. Mapping the recorded signals from different spatiotemporal points of the organoid during cultivation on our device is expected to reveal the electrophysiological development in brain organoids.

Microfluidic devices can be used as powerful analytical tools for biology, ranging from drug discovery to medical diagnostics, requiring only small sample volumes for testing. Such devices enable the scaling down of reaction volumes so that a small amount of material is required and the time for testing is reduced. The ability to perform a suite of diagnostic tests on a single device, with minimal reagents, is a major goal of ‘lab-on-a-chip’ research. For more than 20 years, microfluidic technology has promised to deliver such devices and thereby revolutionize biological and chemical research.

However, the impact of this technology has yet to be fully realized, with plenty of room for innovation^[1]. Currently, the microfluidic channels in most devices play a passive role and are simply used to direct flow. The ability to “functionalize” the channels would impart greater use of these channels, by incorporating functional materials directly within the channels that can partake in, guide, or facilitate the reactions taking place. Integrating functional materials could also serve to introduce new sensing functionalities within the channels themselves. At the same time, rapid prototyping of microfluidic devices is beneficial in a research environment, yet the high cost, slow turnaround, and wasteful nature of the current techniques severely impede the development process. Finally, the lack of portability of the associated detection mechanisms, such as the complex optical microscopes typically employed, makes the ‘lab-on-a-chip’ aspect a moot point. The requirement of complex microscopes and equipment to drive and detect changes within microfluidic channels limits the range of applications of such devices.

We have previously demonstrated the use of Aerosol Jet Printing (AJP) to produce bespoke molds for microfluidic devices, as well as functionalized microfluidic channels^[2]. We showed that such an advanced microscale additive manufacturing method can be used to rapidly design cost-efficient and customized microfluidic devices and add “functional” coatings.

Here, we demonstrate for the first time, printing sensors and active elements within the microfluidic device for electrical detection of protein concentrations. Such functionalized microfluidic biosensors could become a transformative tool in biological and biomedical research, with potential impact in areas such as drug screening, point-of-care diagnostics, and pharmacology. By using an AJP, these sensors can be localized to specific parts of the microfluidic device for constant monitoring and feedback using integrated electrical detection methods, without the need for complex equipment.

[1] G. M. Whitesides, “The origins and the future of microfluidics.,” Nature, vol. 442, no. 7101, pp. 368–73, 2006.
[2] Ćatić, N., et al . (2020). Aerosol-jet printing facilitates the rapid prototyping of microfluidic devices with versatile geometries and precise channel functionalization. Applied Materials Today, 19. https://doi.org/10.1016/j.apmt.2020.100618

Nearly 44 million people worldwide suffer from Alzheimer’s Disease (AD), a progressive, neurodegenerative disease that destroys memories and critical brain functions. AD has become the 6^th leading cause of US deaths and is one of the most expensive condition to care for, costing ~ $300 M/year Diagnosing AD early on and monitoring is key to slow AD’s progression. Very early detection during the Mild Cognitive Impairment (MCI) phase is critical as drug treatments and cognitive therapy are then more effective. Recognizing when MCI is a precursor, leading to AD, is critical as there are no cures when AD fully develops, xonly symptomatic treatments and therapies.

Currently, AD diagnostic methods are very limited. They include first self-reporting or reporting by those close to the patient. This is often inefficient and delayed, in turn delaying treatment past the critical phase of MCI. Once AD is suspected, it can be confirmed by testing cerebro-spinal fluids via spinal tap and complex brain imaging, which is expensive and rarely repeated.

MGO is a biomarker of interest because of the role that MGO, or any MGO-derived advanced glycation end products, play in the pathogenesis of AD. Studies suggest that the glycation of AD-associated proteins, such as amyloid-beta (Aβ) and tau, play a critical role in the pathogenesis of AD³. Semicarbazide-sensitive amine oxidase (SSAO), also known as vascular adhesion protein-1 (VAP-1) is expressed in vascularized tissues, including the brain; furthermore, the presence of SSAO/VAP-1 enzyme has been found in the brains of AD patients⁴. SSAO/VAP-1 immunoreactivity appeared restricted to meningeal and parenchymal blood vessels in the brain. Increased SSAO/VAP-1 immunoreactivity correlates with vascular Aβ deposits in AD patients⁴. SSAO/VAP-1 is responsible for the conversion of proteins into MGO; therefore, MGO could measure vascularized brain tissue, a common symptom of both MCI and AD.

Recently, blood methylglyoxal (MGO) levels were found to correlate significantly with AD¹ and in patients who develop MCI leading to AD². This makes MGO a potential biomarker for early diagnosis of MCI when it is a precursor to AD. MGO levels can potentially differentiate MCI leading to AD from other forms of dementia. Moreover, since MGO is found to increase significantly from MCI to early AD, monitoring MGO levels could measure the impact of drug treatments and cognitive therapy.

Currently, there are no standardized nor portable device on the market to measure MGO, which is a limitation to assess its potential in treating AD. Hence, the present work aims to prototype a novel, hand-held, fast, inexpensive, and accurate Small Volume Blood Diagnostics (SVBD) device, Alz-BioSs™, to test for the presence MCI leading to AD via MGO levels and help monitor the impact of treatments on a regular basis.

Alz-BioSs™ aims to help diagnose and then monitor MCI and AD during the MCI precursor phase to provide patients a greater chance for effective treatments. These patients will also get the opportunity to participate in clinical trials to advance bio-marker research for MCI and AD, while receiving significant medical benefits. Alz-BioSs™ measures MGO in blood plasma by collecting a 0.5 mL blood drop into a single-use microfluidic chip for rapid, passive separation of plasma from blood. Extracted 0.2-0.3 mL plasma reacts then into an adjacent chip coated with bio-reagent o-phenylenediamine (OPD) and gold nanoparticles (AuNP). The resulting colorimetric reaction is detected via a multiple use miniature optical cage system (OCS) using 520 nm LEDs and photodetector s to quantify MGO levels via photo-absorption, akin to some handheld blood glucose monitor used for diabetes monitoring.

[1] Beeri et al., Mech Ageing Dev, 2011
[2] Haddad et al., Internat. J. of Molec. Sci, 2019
[3] Chaudhuri et al., Cell Metab, 2018
[4] Unzeta et al., Internat. J. of Molec. Sci, 2021

Nongenetic optical control of neurons is a powerful technique to study and manipulate the function of the nervous system. Herein we have benchmarked the performance of organic electrolytic photocapacitors (OEPCs) at the level of single mammalian cells. These optoelectronic devices use nontoxic organic pigments that form a planar semiconductor on top of ITO and act as an extracellular stimulation electrode driven by deep red light.
Light stimulation and signal propagation require close contacts between cell membranes and pigments. We could biochemically prove cell viability and show with SEM imaging that cell culture cell lines adhere to the surface and neuronal networks establish and exhibit neurite outgrowth.
Our electrophysiological recordings show that millisecond light-stimulation of OEPCs shifted heterologous expressed voltage-gated K⁺ channel activation by ~ 30 mV. We further demonstrate a time-dependent increase in voltage-gated channel conductivity in response to OEPC stimulation and compared our experimental findings with a mathematical model of this bioelectronic-cell system.
In a further step we cultured primary hippocampal neurons on OEPCs and found that millisecond optical stimuli trigger repetitive action potentials in these neurons. Our findings demonstrate that OEPC devices enable the manipulation of neuronal signaling activities with high precision. OEPCs can therefore be integrated into novel in vitro electrophysiology protocols, and the findings can inspire new in vivo applications for the regeneration of axonal sprouting in damaged neuronal tissues.

Microelectrode arrays (MEAs) play an important role in in vitro electrophysiology, where they support the study of electrogenic tissues. In addition to their use as research tools, they find applications in drug screening and toxicology. A recent trend in the field involves the use of conducting polymer electrodes, such as the commercially available poly(3,4-ethylenedioxythiophene) doped with polystyrene sulfonate (PEDOT:PSS). PEDOT:PSS is a mixed conductor with a capacitance that depends on the volume rather than the area of the film, enabling the fabrication of electrodes with a significantly lower impedance compared to Pt and Au electrodes. I will review the fabrication of these devices and their application in electrophysiology of cell cultures and tissue slices. I will discuss recent work in making them optically transparent and combining them with advanced microscopy techniques.

The interface between biological cells and non-biological materials has profound influences on cellular activities, chronic tissue responses, and ultimately the success of medical implants and bioelectronic devices. The optimal coupling between cells and materials is mainly based on surface interaction, electrical communication and sensing¹.
In the last years, many efforts have been devoted to engineer materials to recapitulate both the environment (i.e., dimensionality, curvature, dynamicity)² and the functionalities (i.e., long and short term synaptic plasticity)³ of the neuronal tissue to ensure a better integration of the bioelectronic platform and cells. In this scenario, resembling also the composition of the neuronal membrane might be beneficial to reconstitute fluidity and proteins’ arrangement (i.e. synaptic receptors) to further optimize the communication between neuronal cells and in vitro bioelectronic platforms².
Here, we explore how supported lipid bilayers(SLBs) can induce different fluidity at the interface and thus modulate the neuronal outgrowth from the polarity phase to synaptic formation. Moreover, those neuronal SLBs have been achieved also on 3D dendritic-like spines to further recapitulate the composition and the peculiar architecture present at synaptic level. Finally, we engineered organic neuromorphic devices with neuronal SLBs to achieve a biohybrid interface with tunable short term plasticity. In turn, this could represent a first step toward in vitro adaptive neurohybrid interfaces to engineering neuronal networks with biomimetic structural and functional connections at synaptic level.
References
1. Lubrano, C. et al. Towards biomimetic electronics that emulate cells. MRS Commun. 10, 398–412 (2020).
2. Mariano, A. et al. Advances in cell-conductive polymer biointerfaces and role of the plasma membrane (2021).
3. Keene, S. T. A biohybrid synapse with neurotransmitter-mediated plasticity. Nat. Mater. 19, 16 (2020).

Supported lipid bilayers (SLBs) are cell-membrane-mimicking platforms of varying biological complexity, that can be formed on solid surfaces and used to characterise the properties of the plasma membrane or to study membrane interactions at the molecular level. The incorporation of microfabricated electrodes and transistors has allowed for their electrochemical characterisation using techniques such as Electrochemical Impedance Spectroscopy (EIS) and transistor-based impedance spectroscopy. In this work, we combine experimental data with numerical simulation to explore the relationship between changes in SLB quality and impedance output, delving into a deeper understanding of the impedance profiles of devices with and without SLB, as well as extracted parameters such as membrane resistance (R_m). We extrapolate this approach to investigate the relationship between microelectrode area and sensor sensitivity to changes in SLB state, towards rational device design. We highlight the trend of electrode size (polymer volume) required for sensing bilayer presence as well as the dependence of the electrode sensitivity to the SLB capacitance and resistance. Finally, we illustrate how our flexible approach of including electrode and transistor measurements to amalgamate characteristic impedance spectra of transistors, overcoming the problem of low frequency noise and errors seen with traditional EIS.

Glioblastoma multiforme (GBM) is one of the most aggressive and invasive brain tumors, thus difficult to eradicate with standard therapies. Indeed, even by combination of multimodal treatments including resection of the tumor, radiotherapy and chemotherapy, most patients experience disease recurrence within one year of diagnosis¹. Use of emerging technologies such as bioelectric therapies is a promising way to improve tumor treatment. These therapies are based on the delivery of pulsed electric fields (PEF) that modifies cell membrane permeability and disrupts the microenvironment of tumors. Despite being very promising, such therapies are not easily applied safely in the brain, as PEFs are mostly delivered by rigid metal electrodes, causing acute and chronic injuries².

Our approach is to develop thin film interdigitated gold electrodes coated with the conductive polymer PEDOT:PSS, as a means of delivering PEFs. First experiments were conducted on glioblastoma cells stably expressing a genetically encoded calcium indicator directly cultured on our in vitro devices, to demonstrate that the delivery of electric pulses induces changes in intracellular calcium and to investigate on the role of ATP signaling in this calcium response. Despite being useful for mechanistic studies at cellular level, 2D in vitro experiments have several limitations as they fail to mimic the microenvironment of tumors. In vivo models are more robust but also expensive, time-consuming and raise ethical issues. For this reason, we developed an intermediate model combining a living organism, a quail embryo, and an in vitro three-dimensional (3D) model of vascularized tumor³. Engineered glioblastoma spheroids were obtained by 3D cell culture and grafted in the chorioallantoic membrane of a quail embryo, which has the particularity to be highly vascularized. At early embryonic stage, tumors are not recognized as foreign bodies due to the lack of immune system, and could even induce its own vascularization by the embryo’s vasculature⁴. Our electrodes were patterned onto a thin layer of the transparent organic parylene-c resulting in a flexible and biocompatible device that can be implanted onto glioblastoma tumors, without damaging surrounding tissue. We demonstrated that PEFs induced the elevation of intracellular calcium in spheroids, as well as the electropermeabilization of cellular membranes and vasoconstriction. This showed that this model is suitable for testing flexible devices under intravital conditions, but also that flexible electronics can be used in standard therapies as a less invasive alternative to rigid metal electrodes. Thanks to their flexibility and biocompatibility, preclinical studies in an in vivo syngenic, orthograft mouse model are now being performed with the same devices, to study effect of PEFs in complete organisms with a functioning immune system.

1. Davis, M. E. Glioblastoma: Overview of Disease and Treatment. Clin J Oncol Nurs 20, S2-8 (2016).
2. Lee, H. Biomechanical analysis of silicon microelectrode-induced strain in the brain. J. Neural Eng. 2, 81–89 (2005).
3. Lefevre, M. C. Integrating flexible electronics for pulsed electric field delivery in a vascularized 3D glioblastoma model. npj Flexible Electronics 9 (2021).
4. Ribatti, D. Chapter 5 Chick Embryo Chorioallantoic Membrane as a Useful Tool to Study Angiogenesis. in International Review of Cell and Molecular Biology vol. 270 181–224 (Academic Press, 2008).

PEDOT:PSS has a proven record as an electrode coating material for biological applications. One of the key advantages is its volumetric coupling with electrolytes which enhances the ionic-electronic transduction, a property that reduces the electrode impedance and increases the charge injection capacity. Despite significant progress, the operation mechanisms of PEDOT:PSS remains to be fully understood. Especially, few reports exist on the electrical stimulation properties of PEDOT:PSS, establishing a research topic that deserves more attention.
In this work, we investigated the electrical stimulation performance of PEDOT:PSS-coated electrodes in the context of electroporation, the phenomena where the cell membrane is permeabilized by means of electrical stimulation. Typically electroporation is performed with high amplitude, voltage-controlled, monophasic pulses.
We show that the PEDOT:PSS coating thickness has an important influence on the stimulation performance. Besides increasing the electrode capacitance, thicker coatings can endure more stimulation pulses and withstand higher voltages without loss of performance, thus enhancing the lifetime of the electrode. Coating electrodes with PEDOT:PSS strongly reduces irreversible electrochemical reactions that otherwise lead to changes in pH and the generation of reactive oxygen species. Coated electrodes exhibit a slightly higher threshold for cell stimulation, however, little electropermeabilization is observed at voltages that greatly exceed the threshold for uncoated electrodes.
The results demonstrate some of the remarkable stimulation properties of PEDOT:PSS and emphasize the importance of electrode material for electrical stimulation applications.

Interfacing biomimetic membranes with electronic devices promises to expand our understanding of the function of these membranes in disease states. The challenge is to form supported lipid bilayer (SLBs) on (semi)conducting films while sustaining high sensitivity and operational stability. In this talk, I will show the traits of n-type organic semiconductors for such biological interfaces. I will show an ion channel sensor based on an n-type microscale organic electrochemical transistor (OECT), which interfaces an SLB embedded with a pore-forming protein. The polymer is particularly sensitive to divalent cations, endowing the system with biomimetic properties. In another platform, a similar polymer forms the basis of a biocatalytic hybrid that detects minute changes in metabolite concentrations. The side-chain engineering of semiconducting polymers prompts the development of next-generation bioelectronic hybrids where electron-transporting polymers establish bilateral electronic communication with living systems.

Membrane active compounds such as antimicrobial peptides, ionophores and voltage sensitive dyes have garnered attentions due to their interesting mode of actions and their potential applications in biotechnology and drug development. The creation and the improvement of such molecules are often hindered by a lack of analytical tools to screen molecules in a high throughput way or to study their fundamental mechanism in membranes. Our group has been developing different configurations of cell-free electrophysiological tools for the characterization of biomolecule-membrane interactions. In this work, we will present our recent setup, lateral black lipid membrane, with a special focus on the challenges we encountered during the development. Taking an advantage of the home-made tool, we have discovered a unique cooperative function between two well-known antimicrobial peptides (LL-37/HNP1) that kills bacteria more efficiently while minimizing the host damage by suppressing mammalian cell membrane lysis, named “antimicrobial peptide double cooperativity”. Such a double cooperativity may be used in our immune system and may help with developing efficient and safe antimicrobial agents in the future.

Dynamic chemical processes at cell surfaces including exocytosis of hormones and neurotransmitters play important roles in chemical signaling and intracellular communication. The ability to detect and quantify these events with high spatial resolution holds the key to understanding many biological pathways and physiological functions. Due to a lack of optical activity, electrochemical techniques such as amperometry and fast-scan cyclic voltammetry are routinely used to detect compounds released from cells with high spatial and temporal resolution. However, only a limited number of desired analytes are electroactive. To overcome this limitation, we have developed a series of electrophysiological probes based on ligand gated ion channels reconstituted into stabilized lipid membranes. Signal is transduced by ligand-protein binding between target analytes and ion channels embedded in the lipid membranes to generate ion flux that can be measured using traditional electrophysiology methods. The resulting sensor provides sensitive, selective, and label-free detection of analytes with high temporal and spatial resolution. When combined with high resolution topographical imaging using scanning probe microscopy, high resolution chemical imaging may be possible. This presentation will focus on the preparation of the stabilized membranes, the ion channel probe sensors and recent efforts to couple this approach to scanning probe microscopy to generate high resolution chemical maps of living cells and tissues.

The merging of electronics with biology at the nanoscale gives rise to new opportunities to modulate cellular behaviour. We present an assertion that nano-bipolar electrode technology can play a part in this by enabling methods to modulate cellular-electrical communication. To date, nanobipolar electrodes have not been utilised in cells. In a first, we present an impedimetric method and fluorescent data that demonstrate nano-bipolar electrodes placed within an electric field become polarised enabling intracellular based redox reactions to be modulated on-demand. We also now know faradaic bioelectrical circuits including trans plasma membrane electron transport underpin cell dysfunction. Therefore, the ability to control such circuits may give rise to new ways to control underlying biology and disease. Towards this vision, we use membrane-embedded carbon nanotube (CNTs) as bipolar nanoelectrodes and show we can control electron flow with externally applied electric fields across biological membranes. New mechanistic insight into this newly described phenomenon at the nanoscale is given. The results that will discuss give rise to a new method to modulate cell behaviour via wireless control of electron transfer.

Materials science in general, and nanoscience and nanotechnology, in particular, have promised multiple applications enabled by an unprecedented control of matter. One of them is the ultimate miniaturization of electronic components and circuits to the scale of individual molecules. This exciting field is known as Molecular Electronics¹. Shortly after
opening the door for reliable single-molecule conductance measurements², biomolecules were also considered as the subject of these studies³. The field of biomolecular electronics has experienced a boom in the last decades. Not only the developments in microscopy have allowed researchers to study biological details of fundamental processes with a high spatial and temporal resolution⁴, but nanoscience tools such as Scanning Probe Microscopies have led to biophysical studies at the single-molecule level. Notably, Scanning Tunneling Microscopy (STM)⁵ has enabled researchers to perform single-molecule electronic studies of biomolecules³, including nucleic acids⁶, redox proteins⁷, and more complex biosystems⁸.

In this contribution, we show examples of the new biomolecular electronics projects in our lab (http://faculty.uml.edu/artes), where we integrate classical nanotechnology methods from materials science and characterization for the study of different
biological systems:

#The analysis of the biomolecular interactions at the RNA Induced Silencing Complex.
#The single-molecule electrical detection of circulating tumor nucleic acids to study cancer biomarkers and the early diagnostics through liquid biopsy.
We are currently obtaining preliminary results on the charge transport through double-stranded⁹ and single-stranded RNA oligonucleotides¹⁰, showing that these single-biomolecule electrical fingerprints contain important biophysical information. These methods and results could be applied to numerous biological problems, paving the way to a whole new body of biophysics and materials science knowledge. In particular, electrical signals have demonstrated helpful in detecting RNA. This, combined with new results from biomolecular interactions, could allow a complete molecular understanding of how regulatory RNA elements work. Also, this knowledge could pave the way for the design of next-generation single-molecule nanobiosensors¹¹ for biomedicine and public health monitoring.

REFERENCES
1. Molecular rectifiers. A Aviram, MA Ratner. Chemical physics letters, 1974
2. Measurement of single-molecule resistance by repeated formation of molecular junctions. B Xu, NJ Tao. Science, 2003
3. RNA BioMolecular Electronics: towards new tools for biophysics and biomedicine. Arachchillage KG, Chandra S, Piso A, Qattan T, Artes Vivancos JM. Journal of Materials Chemistry B, 2021
4. Single-molecule spectroscopy, imaging, and photocontrol: foundations for super-resolution microscopy. WE Moerner. Angewandte Chemie International Edition, 2015
5. Scanning tunneling microscopy. G Binnig, H Rohrer. Surface Science, 1983
6. Direct conductance measurement of single DNA molecules in aqueous solution. B Xu, P Zhang, X Li, NJ Tao. Nano letters, 2004
7. Transistor-like behavior of single metalloprotein junctions. JM Artés Vivancos, I Díez-Pérez, P Gorostiza. Nano letters, 2012
8. Probing Bioelectronic Connections Using Streptavidin Molecules with Modified Valency. B Zhang, E Ryan, X Wang, S Lindsay. Journal of the American Chemical Society 2021
9. Single-molecule conductance of double-stranded RNA oligonucleotides.S Chandra, KGGP Arachchillage, E Kliuchnikov, F Maksudov, S Ayoub,V Barsegov, and JM Artes Vivancos. Nanoscale. In revision
10. Charge-transport through pi-stacked bases in single-stranded RNA. S Chandra, KGGP Arachchillage, E Kliuchnikov, F Maksudov, V Barsegov, and JM Artes Vivancos. In preparation
11. Detection and identification of genetic material via single-molecule conductance. Li, Y., Artés Vivancos, J.M., Demir, B., Gokce, S., Mohammad, H.M., Alangari, M., Anantram, M.P., Oren, E.E. and J Hihath. Nat Nano. 2018

The ability to control and direct specific ion transport across multiple scales is one of the fundamental challenges for bioelectronic applications. While researchers have traditionally focused on organic materials such as conductive polymers for these tasks, recent progress in materials science associated with development of 1D and 2D nanomaterials is beginning to offer potential alternatives. In particular, precise control over molecular confinement, readily achievable in these materials, provides unique opportunities to enhance their performance. I will discuss several examples of controlling ion transport in highly confined 1D carbon nanotube porins and 2D channels in layered materials. I will also highlight some of the unusual mechanisms that enhance ion transport efficiency and ion transport selectivity in these systems, discuss the energy barriers for transport in these systems, and outline some challenges faced in translating these unique transport properties into applications.

Despite technological advances in biomolecule detections, evaluation of molecular interactions via potentiometric devices under ion-enriched solutions has remained a long-standing problem. To avoid severe performance degradation of bioelectronics by ionic screening effects, we cover probe surfaces of field effect transistors with a single film of the supported lipid bilayer, and realize respectable potentiometric signals from receptor–ligand bindings irrespective of ionic strength of bulky solutions by placing an ion-free water layer underneath the supported lipid bilayer. High-energy X-ray reflectometry together with the circuit analysis
and molecular dynamics simulation discovered biochemical findings that effective electrical signals dominantly originated from the sub-nanoscale conformational change of lipids in the course of receptor–ligand bindings. Beyond thorough analysis on the underlying mechanism at the molecular level, the proposed supported lipid bilayer-field effect transistor platform ensures the world-record level of sensitivity in molecular detection with excellent reproducibility regardless of molecular charges and environmental ionic conditions.

The field of bioelectronics has developed rapidly in recent years and has had a huge impact on healthcare and fundamental biological study, providing new tools for studying cells and driving unprecedent approaches for monitoring human body.[1] Wireless electronic system is a essential part of bioelectronics to modulate the chemistry inside a cell without piercing the plasma membrane of the cell, which could hinder normal physiological activity of the cell.[2] In this work, we have taken different wireless communication strategies to assess the electric field inside a cell with the model drug delivery system with polypyrrole for the first time to shed light on communication with complex biological environments to realize intracellular sensors and actuators.
First, we have built upon a drug delivery system with electropolymerized polypyrrole to incorporate fluorescein, a model drug whose release can be measured by fluorescence.[3] The drug release and leakage profile with potentiostatic and potentiodynamic actuation are investigated based on their compatibility with wireless communication strategies. Using a three-electrode measurement setup, we demonstrate a 3.8x (2.6x) increase in fluorescein release with potentiostatic (potentiodynamic) method compared to the non-actuated sample. Further, the impedance of polypyrrole was measured with various DC voltages to address the conductivity and redox status of the polymer in different electric fields, which shows lowest charge transfer resistance of 92 ohm at 0.5 V vs. Ag/AgCl at the oxidized form of polypyrrole from the range of -0.5 V to 0.5 V vs. Ag/AgCl. Based on the impedance measurements, we project the performance of polypyrrole under external electric fields.
It is critical to understand the impedance components of an experimental environment and a cell in order to effectively communicate wirelessly with it.[4] To this end, we have simulated the electric field inside a cell assuming a chamber with 5mm diameter that is adaptable for live-cell imaging, with potentiostatic and potentiodynamic actuation strategies, using Ansys Maxwell. From the simulation, we have optimized the configuration of internal and external electrodes as well as the best way to maximize the tolerance of mismatch between those two electrodes pair. By measuring the impedance of the system in different physiological solution, the electric field profile and circuit components were extracted to further reinforce the simulation.
A microchip with 100 micrometer diameter is fabricated using conventional optical lithography and injected into an amoeba cell to check its toxicity and normal physiological behavior over the course of 12 hours under the continuous live cell imaging. After confirming the biocompatibility of the microchip, we implement the drug delivery system on-chip by depositing polypyrrole and testing its drug release profile with potentiostatic method, inspired by bipolar electrochemistry, and potentiodynamic method.
In summary, we have compared two different wireless actuation methods and their impact on drug release profile with a model drug delivery system using polypyrrole and fluorescein, revealing the redox behavior of polypyrrole in different electric field and the distribution of electric field from the external stimulus. The behavior of an amoeba cell with a chip inside is analyzed using continuous live cell imaging. Being able to release a drug that trigger intracellular signaling pathways from a chip inside a cell will open a new possibility to control endogenous cellular system wirelessly, which we are aiming to realize as a next step.
1. T. Someya et al., Nature, 540, 379 (2016)
2. G. He et al., Adv. Funct. Mater., 30, 1909890 (2020)
3. S. Miar et al., Front. Chem., 9, 599631 (2021)
4. T. Taghian et al., J. R. Soc. Interface., 12, 20150153 (2015)

Transmembrane proteins (TMPs) are Nature’s biorecognition, sensing, and transduction elements and critical components in biomimetic sensing. However, they are among the most challenging biomolecules to integrate into biosensors because they require a lipid bilayer to remain functional. Successful bilayer and TMP integration with bioelectronic devices would enable new capabilities in in vitro biosensing and biological actuation. To date, reconstitution of lipid bilayers onto sensing surfaces requires significant optimization of the abiotic/biotic interface. Nonetheless, current hybrid biotic-abiotic devices show great promise in biosensing, but even more so if arrays of TMPs tailored to sense specific targets could be produced. Such scale up is necessary for rapidly identifying the most sensitive protein sensors, providing a myriad of sensing elements that can monitor a variety of targets simultaneously, or producing enough data to enable machine learning approaches that can extend beyond biosensing into bioanalytical applications.
Our team has developed several approaches to integrate TMPs into supported lipid bilayers that functionalize organic bioelectronic devices. In this presentation, I will cover two methods. First, I will describe a technique to harvest membrane vesicles from live cells and using them to coat a surface of a bioelectronic device. Once formed, they serve as an authentic replica of the plasma membrane from a variety of cell types. For the second approach, we use cell-free synthesis of proteins, in which cellular extracts are used to synthesize transmembrane proteins directly into lipid bilayers that coat the sensing surface. I will describe several sensing applications using both approaches.
Our eventual goal is to build TMPs into arrays of biomembranes on bioelectronic devices that sense their functions and activity levels using the dual modality of electronic and optical means. The criticality of dual mode data collection is that, for transporters and ion channels, electrical means can be used label-free to read out flux of material across the membrane. Optical approaches offer the possibility to simultaneously obtain confirmation of folding with protein folding reporters, as well as obtaining structural information. Combining all these features into one platform offers a wealth of possibilities for many biosensing and bioanalytical applications and could be used to understand how the properties of the membrane influence the activity of TMPs.

Intracellular cargo delivery is a critical and challenging step in establishing direct bio-inorganic communication. Silicon nanowire arrays, utilizing high aspect ratio dimensions, have emerged as a powerful platform for electrically connecting the intracellular space to external devices. We have developed and characterized a conductive polypyrrole-nanowire device capable of storing molecular cargo for electrically triggered intracellular delivery. Fluorescent cargos, doped in electroresponsive polypyrrole matrices at wire tips as well as entire NW arrays, were released into endothelial cells upon polypyrrole reduction. The combined polymer carrier and nanowire architecture demonstrated comparable delivery kinetics to transfer from solution while requiring an order of magnitude less cargo loading. This hybrid polymer-semiconductor platform extends methods available for intracellular delivery and links electrical signaling from artificial systems with living molecular transduction.

Organic electrochemical transistors (OECT) based on the conductive polymer PEDOT:PSS have been used in different organisms to study and analyze different biological responses. The main advantages of the OECTs are the combination of high quality electrical signals and optical monitoring due to the transparency of PEDOT:PSS thin films. We decided to use these properties to study plant membrane proteins because, even though plant transport mechanisms have been studied for many years, the study of individual proteins is still a big challenge. One of these proteins is the copper transporter 1 (COPT1), which maintains the plants' copper homeostasis, but its function has not been totally characterized. Therefore, we formed a hybrid supported lipid bilayer (SLB) with membranes from transient GFP and GFP-COPT1-transfected Arabidopsis thaliana mesophyll protoplasts and fusogenic liposomes on top of PEDOT:PSS. SLB formation and fluidity were determined by fluorescence recovery after photobleaching following the diffusion coefficient calculation. Both samples were full photobleaching recovered and had similar diffusion coefficients, confirming the high quality of SLBs. Next, we combined SLBs with OECTs, and using electrochemical impedance spectroscopy (EIS) measurements, we showed that plant SLB formed a layer that blocks the passage of ions from the electrolyte into the conductive polymer, increasing the system's impedance. We used an equivalent electrical circuit modeling to extract membrane resistance (R_m) from the raw impedance. Then, we tested these SLBs with different CuSO₄ concentrations ranging from 0-100 µM. We observed a decrease in the R_m in both cases, but it was higher in those overexpressing COPT1, indicating that in the presence of copper, COPT1 transporter is open. Our platform can be used to analyze profoundly COPT1 transporter mechanism and adapted to investigate more membrane proteins involved in transport mechanisms in plants.

Photosynthesis, the process by which solar energy is harvested and converted to biomass by plants, algae and cyanobacteria, consists of efficient machineries for light harvesting and catalysis. However, photosynthesis is a sum of several processes and overall inefficient for biomass conversion. Scientists have been trying to re-direct the photosynthetic electron transport pathways at various points using a multitude of strategies to mitigate bottlenecks or enable novel chemistries. It was accepted that electron re-direction at picosecond timepoints after light harvesting would not be possible since these early events occur at inaccessible sites buried deep within the photosystem protein complexes. Here, we show using ultrafast transient absorption spectroscopy that electron re-direction on the sub-picosecond timescale is possible in isolated photosystems and living photosynthetic bacteria. Indeed, the early charge re-direction occurs in the presence of the electron mediator 2,6-dichlorobenzoquinone, which was originally believed to extract charge only at a later time point (the plastoquinone sites) within photosystem II. We reveal that charge extraction occurs at the chlorophyll pigments, which are in resonance with the primary donors when photoexcited. This work expands our understanding of energy transfer processes within reaction center complexes and in photosynthesis more generally; it also heralds a new paradigm of how photosynthesis can be re-wired, for example in contexts such as bioenergy and artificial photosynthesis.
Here, we demonstrate using in vivo ultrafast transient absorption spectroscopy that it is possible to extract electrons directly from photoexcited PSI and PSII using exogenous electron mediators, such as DCBQ. These results highlight that photosynthesis may be rewired at sub-ps timescales, at the earliest possible step in the photosynthetic electron transport chain (PETC) chain.

Behavioral evidence suggests that the earth magnetic field is used by many different species as a means to orient themselves when traveling over long distances. However, certainty about the underlying magnetic field sensing mechanism remains elusive. As one of the leading theories, the cryptochrome hypothesis stipulates that the redox cofactor flavin is involved in the sensing mechanism: after being exposed to blue light, flavin and tryptophan form a radical pair whose state is dependent on the magnetic field present.
Since radical pairs can be formed by an electric field, we examined the sensitivity of flavin mononucleotide (FMN) solutions to magnetic field influence in an electrochemical cell as an example for redox cofactors in general.¹ We designed two three-electrode electrochemical cells that could be integrated into a magnetic field apparatus able to generate fields up to 1T. We observed that the redox currents depend strongly on applied magnetic fields. We then showed that magnetic field sensitivity in redox currents is a function of setup geometry and additionally relies on FMN existence as an unbound species in solution.
We then investigated the mechanisms underlying the observed current enhancements in the magnetic field. The scan rate dependency of the peak current as well as control experiments excluding proposed radical pairs and the inclusion of a radical quencher pointed towards the main cause of the magnetic field sensitivity being the magnetohydrodynamic effect. These findings were further supported by simulations showing the influence of magnetic fields on the current of free radicals in solution via the Lorentz force. Experiments with other redox cofactors further corroborated our conclusion that the magnetohydrodynamic effect is the dominant mechanism of magnetically-enhanced redox currents in solutions of cofactors.
This work delivers an experimental platform for careful investigation of the role of magnetic field in redox reactions, as well as offers practical means to modulate rates of cofactor redox reactions in engineering applications.
1. Park*, Koehler*, Varnavides, Anikeeva, Angew. Chem. Int. Ed. 2021. * - equal contribution

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants are of particular interest because they can potentially increase the virulence and transmissibility of coronavirus disease 2019 (COVID-19) or reduce the effectiveness of vaccines. In this aspect, a strategy to rapidly screen SARS-CoV-2 variants with large testing capacity is in urgent need. However, screening SARS-CoV-2 variants is a challenge because the target of biosensors can mutate.
We conceived the idea of variant screening from the fact that all known SARS-CoV-2 variants share the same virus receptor, angiotensin-converting enzyme 2 (ACE2). Notably, variants with high transmissibility show higher binding affinity with ACE-2. In this study, we fabricated a portable electrical biosensor for SARS-CoV-2 variant screening. To optimize the biosensor, we introduced the synthetic virus that mimics important features of SARS-CoV-2, such as the bilayer structure, size, and composition.
The developed biosensor successfully detected inactivated SARS-CoV-2 in 20 min and showed comparable sensitivity to molecular diagnostic tests (~165 copies/mL). Our results indicate that i) synthetic virus can be a useful approach to model and optimize biosensor and ii) a virus receptor-based biosensor can be an effective strategy for screening infectious diseases to prevent pandemics.

Biocompatible hydrogels are of great interest in the field of biomedical research and translational medicine. Hydrogels are used for encapsulation of therapeutic or biological agents such as small molecule drugs, proteins, or biomolecules. In this work, we present a new method integrating printing processes to fabricate biocompatible electronic hydrogels. The electronic gels are customized to offer different functionalities for bio-devices including supercapacitors, electrodes, and Organic Electrochemical Transistors (OECT). The stretchable jelly devices exhibit high conductivity, high transconductance (in the mS range), and self-healing properties. The biocompatible functionalized jelly transistors were successfully implanted in plant tissue and data recording the ionic activity was collected for over a week. Our gel-based electronics are excellent candidates for tissue engineering scaffolds, contact lenses, and drugs eluting systems. This work paves the way for the integration of electronics gel systems with in-vivo tissue models for monitoring biological activity.

Organic electrochemical transistors (OECTs) have been widely used as biosensors to sense biomarkers such as pH, ions, small molecules (glucose, lactate), nucleic acids, and even proteins and cells in last few decades. Various bio-recognition elements like ion-selective membranes (ISM), enzymes, aptamers, and antibodies have been integrated with OECTs for sensing. Among those bio-recognition elements, aptamers have drawn a lot of attention owing to their ease of chemical synthesis, wider applicability of different targets, good thermal/environment stability and low-cost. Electrochemical aptamer-based sensors based on a single modified electrode has been used as a standard structure in the aptamer-based biosensor research community. The sensing ability is achieved by monitoring the change of electron transfer rate in redox reporter/electrode interface that is altered by the structural rearrangements of aptamers induced by target binding. However, the current change in this sensor is usually very small especially when the area of sensing electrode is reduced for miniaturization purposes, which limit its sensitivity. In this work, we use the OECT as an on-site amplifier to enhance this small current change in the surface of an individual electrode. A new device structure is proposed that can be used to perform OECT testing and traditional electrochemical measurements like cyclic voltammetry (CV) and square-wave voltammetry (SWV) at the same time. Au working electrode functionalized by methylene-blue modified aptamer, PEDOT:PSS counter electrode and on-chip Ag/AgCl reference electrode are monolithically integrated in a same substrate. During the traditional electrochemical measurement with three-electrode setup, the change of in-plane conductivity at the counter electrode (PEDOT:PSS) can be monitored at the same time and can be regarded as the output of an OECT device. By using this approach, direct amplification of the current in the working electrode (gate) to the in-plane current in the counter electrode (channel) can be achieved. The device can be used to sense transforming growth factor beta 1 (TGF-β₁), which is an important cytokine of interest in wound healing. A sensitivity enhancement around 3000-fold can be observed in our device (294 μA/dec) compared to the sensing results from a single electrode-based sensor (85 nA/dec), with similar detection limit (~1ng/mL). This new approach is likely to be generalizable to a wide range of electrochemical aptamer-based sensors to enhance the sensitivity, which can help to ease the burden in the backend signal amplification.

Delayed administration of appropriate antibiotics for the treatment of infections greatly reduces the chances of survival. Therefore, it is necessary to prescribe effective antibiotics in a timely manner to avoid misuse of antibiotics through a rapid antibiotic susceptibility test (AST). However, conventional AST methods involve blood culture, pure culture and susceptibility testing. Due to series of steps, the methods require more than three days. Here, we report a vertical capacitance sensor functionalized with aptamers that can detect bacterial growth and perform AST faster. Due to the heaviness of blood cells, they sunk by gravity and the influence on capacitance was reduces in vertical structure. Thus, bacterial growth in blood was monitored in real-time by measuring changes of capacitance at f = 10 kHz. Moreover, information on biofilm formation could be obtained by the measurements at f = 0.5 kHz during blood cultures. Bacterial growth and biofilms formation were inhibited above the minimal inhibitory concentration of antibiotics. Therefore, the novel vertical aptamer immobilized capacitance sensor could be applied to rapid AST from positive blood cultures without the other culture processes.

O₂ reduction is the key reaction in cellular respiration. Respiration pathways also produce reactive oxygen species (ROS) as byproducts: peroxide, superoxide, and hydroxyl radicals. The balance of these species is critical to homeostasis, as high levels can result in toxic effects, while lower concentrations mediate important signaling pathways. Artificial manipulation of oxygen chemistry in biological systems using electrical devices could be a powerful tool for basic research and eventually biomedical devices. H₂O₂ plays a significant role in a vast range of physiological processes in aerobic environments where it is produced by cells and performs vital tasks in redox signaling. The sensitivity of many biological signaling pathways to H₂O₂ opens up a unique direction in the development of bioelectronics devices to control levels of oxygen and reactive-oxygen species (ROS). Here we present microfabricated devices that can modulate local oxygen concentrations between saturation, 21%, and complete hypoxia, as well as devices that can additional reduce oxygen selectively to hydrogen peroxide. This allows precise generation of hypoxic and/or ROS gradients. We discuss these findings in the context of existing neuromodulation electrodes and protocols, as well as demonstrations of successfully modulating human H₂O₂-sensitive Kv7.2/7.3 (M-type) channels expressed in in vitro single-cell models.

Thin film transistors are ubiquitous from displays to detectors. However, their use in biosensing is limited by a lack of techniques to functionalise the transistors. In this talk, the foundation of employing them as cost-effective portable biosensors by modifying the surface functionality will be discussed. One common functionalisation method is based on dropcasting which is to modify the whole chip surface with organofunctional molecules and antibody for the immobilisation of the specific biomolecule. But this method normally allows only one type of biomolecules to be detected at one time due to the spread of the molecule droplet over the sample surface. In this study, we use additive electrohydrodynamic printing to locally functionalise thin film transistors. The local functionalisation is achieved by printing features using microscale nozzles, enabling different surface functionalities on each transistor. This presents a unique method to create portable biosensors that are able to detect more than one biomolecule simultaneously, facilitating the diagnosis of human diseases.

Glucose bio-sensing technologies have received increasing attention in the last decades, primarily due to the fundamental role that glucose metabolism plays in diseases (e.g. diabetes). Molecularly imprinted polymers (MIPs) could offer an alternative means of analysis to a field that is traditionally dominated by enzyme-based devices, posing superior chemical stability, cost-effectiveness and ease of fabrication. Their integration into sensing devices as recognition elements has been extensively studied with different readout methods such as quartz-crystal microbalance (QCM) or impedance spectroscopy. In this work, a dummy imprinting approach is introduced, describing the synthesis and optimizaion of a MIP towards the sensing of glucose. Integration of this polymer into a thermally conductive receptor layer was achieved by micro-contact deposition. In essence, the MIP particles are pressed into a polyvinyl chloride (PVC) adhesive layer using a polydimethylsiloxane (PDMS) stamp. The prepared layer is then evaluated with the so-called heat transfer method (HTM) methodology, allowing the determination of the specificity and sensitivity of the receptor layer. Furthermore, the selectivity was assessed by analyzing the thermal response after infusions with increasing concentrations of different saccharide analogues in phosphate buffered saline. The obtained results show a linear range of the sensor of 0.0194-0.3300 mM for the detection of glucose in phosphate buffered saline. Finally, a potential application of the sensor was demonstrated by exposing the receptor layer to increasing concentrations of glucose in human urine samples, demonstrating a linear range of 0.0444-0.3300 mM. The results obtained in this paper, highlight the applicability of the sensor both in terms of non-invasive glucose monitoring and for the analysis of food samples.

This talk will present how our merger of organic bioelectronics and bacterial infection biology has opened exciting, new opportunities for in vitro studies in the lab as well as for infection diagnostics and control. Our approach takes advantage of the electro-catalytical properties of conductive polymers for mutual communication between bioactive surfaces and bacterial cells growing in liquid culture and as biofilms. Biofilm forms when free-living planktonic bacteria undergo a phenotypic transition, producing dense bacterial aggregates embedded in extracellular matrix. We take advantage of the dual organic-conductive nature of electrically conducting oligomers and polymers to develop novel materials and strategies to control biofilm formation and diagnose infections. Based on the conducting polymer poly(3,4-ethylenedioxythiophene (PEDOT), we show how PEDOT can be used for rapid and sensitive potentiometric sensing of bacteria in liquid cultures via their secreted redox active compounds, and how an organic electrochemical transistor (OECT) can be used to monitor growth of Salmonella cultures in real-time. We also show that PEDOT can act as an electron mediator for bacterial metabolism, demonstrating how growth of Salmonella biofilm can be modulated by the electrochemical state of the polymer. By functionalizing PEDOT with biocide agents, the material showed efficient antimicrobial activity as a surface coating. This was illustrated in PEDOT functionalized with silver nanoparticles (AgNP), where a synergistic effect of AgNP and the electrical input nearly completely prevented growth of Staphylococcus aureus. To overcome the lack of methods for specific reporting of the biofilm lifestyle, we developed methods for specific detection of extracellular matrix components. Utilising the opto-electronic nature of conjugated oligothiophenes, we established ‘optotracing’ as a non-toxic, fluorescence-based method for identification of ECM components, such as amyloid curli fibers and cellulose, in liquid cultures as well as when biofilm forms at the air-solid interface in real-time. Optotracing was further applied in a culture-independent diagnostic assay for biofilm-related urinary tract infection caused by uropathogenic E. coli, and as an algorithm-based optical sensing method specific for Staphylococcus aureus. We are now extending the applicability of optotracing technology for automated biofilm diagnostics and point-of-care bacteria identification.

Current methods of cell culture are extremely labour intensive. Cells must be harvested, plated, fed, grown, passaged, counted and replated. Only then may they be tested. Once the relevant stimuli has been applied they must then be imaged or counted to determine the effect of such a stimuli. Factor in all the control tests and the repeats needed to demonstrate reliability and it quickly becomes an enormous task.
To help solve this problem, we have developed a platform to enable continuous cell monitoring via impedance spectroscopy. Most impedance spectroscopy is done through bare electrodes made of an inert metal, usually gold.^[1,2] Our platform instead utilises a biocompatible polymer surface,^[3,4] above embedded silver electrodes to constantly monitor a cell culture, without the need for manual counting or imaging. This not only reduces the time requirement of an experiment, but it also allows more data to be collected, particularly where an applied stimuli has a real time response. The platform has successfully demonstrated that it may effectively differentiate not only between the presence or absence of cells on the surface, but also their relative densities. Future refinements may improve the sensitivity of the platform further, or even probe single cell behaviour.

[1] T. Chalklen, Q. Jing, S. KarNarayan, Sensors 2020, 20, 5605, 10.3390/s20195605.
[2] S. Khoshfetrat Pakazad, A. Savov, A. van de Stolpe, R. Dekker, J. Micromechanics Microengineering 2014, 24, 034003, 10.1088/0960-1317/24/3/034003.
[3] M. Smith, C. Lindackers, K. McCarthy, S. KarNarayan, Macromol. Mater. Eng. 2019, 304, 1800607, 10.1002/mame.201800607.
[4] M. Smith, T. Chalklen, C. Lindackers, Y. Calahorra, C. Howe, A. Tamboli, D. V. Bax, D. J. Barrett, R. E. Cameron, S. M. Best, S. KarNarayan, ACS Appl. Bio Mater. 2020, 3, 2140, 10.1021/acsabm.0c00012.

The monitoring of dissolved oxygen is a key parameter in many fields, namely the treatment and monitoring of various cerebral traumas. Leveraging complementary metal oxide semiconductor (CMOS) manufacturing techniques and biocompatible electrolyte polymers, electrochemical sensors hold the potential for compact, simple, and scalable point-of-care dissolved oxygen sensors. We present here a discussion of guiding microelectrode design principles based on a characterization comparison study conducted across multiple Clark sensors with varying microelectrode geometries. Geometries were covered with a biocompatible Nafion polymer membrane. Polarographic sweeps were conducted on various microelectrode topologies while monitoring devices’ sensing current responses across a 50.3mmHg change in dissolved oxygen in deionized aqueous solution. An optimum design with a ~7-fold improvement in sensitivity was achieved and the results validated with theory.

Photosynthesis relies on the ‘light’ reaction, carried at by the photosynthetic electron transport chain (PETC), to generate energy carriers for use by the ‘dark’ reaction to carry out reactions such as carbon dioxide fixation. We now have the capability to electrochemically tap into the PETC in vivo and harness its electrons for fundamental studies and for alternative energy conversion technologies.¹ However, enormous hurdles remain when wiring living photosynthetic cells to electrodes compared to the wiring of molecular catalysts or enzymes. Endogenous mediators are typically required to aid electrochemical wiring, but their use comes with an energetic cost, and their mode of intracellular interaction is not well understood. The electrode structure itself also require careful consideration to ensure optimal energy and electron transfer at the bio-electrode interface.
Here, I will talk about our latest efforts to understand how we can better electrochemically tap into the PETC in vivo. Specially, I will describe our efforts to understand i) light-induced exo-electrogenesis by cyanobacteria; ii) diffusional electron shuttles targeting the PETC,² and iii) how the electrode architecture affects the wiring of photosynthetic cells to electrodes.³
References
1. Zhang, J. Z. & Reisner, E., (2020) Advancing photosystem II photoelectrochemistry for semi-artificial photosynthesis. Nature Rev. Chem., 4 , 6-21.
2. Clifford, E. R., Bradley, R. W., Wey, L. T., Lawrence, J. M., Chen, X., Howe, C. J. & Zhang, J. Z., (2021) Phenazines as model low-midpoint potential electron shuttles for photosynthetic bioelectrochemical systems. Chem. Sci., 12, 3328 - 3338.
3. Chen, X., Lawrence, J. M., Wey, L. T., Schertel, L., Jing, Q., Vignolini, S., Howe, C., Kar-Narayan, S., Zhang, J. Z., 3D-printed hierarchical pillar array electrodes for high performance semi-artificial photosynthesis, https://doi.org/10.33774/chemrxiv-2021-rrg7q-v2

Organic bioelectronics go wild in the past decades. As the flagship device, organic electrochemical transistors (OECTs) provide a new choice for next-generation bioelectronic devices1. For one thing, inherent advantages of organic electronics get retained in OECT, such as the diversity of material selection and tunability of structure design. For another, the nature of volumetric capacitance and high transconductance lead to exceptional signal amplification.
However, despite past trials to promote OECT applications in the bioelectronic field, there is still an important issue: low mechanical stretchability2. Most conventional electronics are rigid and stiff. The significant modulus mismatch causes physical incompatibility with soft and elastic biological interfaces, triggering device failures under repeated deformation like bending, twisting, or beating.
As an essential category of organic electronics, OECT has been studied for decades. However, soft and stretchable OECT is still in its infancy. Previous research is limited, and there are no satisfactory devices currently for practical usage. The core problem is maintaining the efficient electrical performance of original OECTs when improving their mechanical strength, as strain-induced deformation tends to influence two sides adversely. To achieve trade-offs, it’s imperative to select an appropriate material system.
In this talk, I will first backtrack the conceptualization of stretchable OECTs2. Former developments of relevant areas, including the OECT itself and other stretchable electronics, build fundamental frameworks for the direction. Then, I will show how stretchable conductors3, soft and healable conducting polymers3,4, and stretchable iontronic hydrogels5 advance the development of stretchable OECTs. Finally, I detail how soft organic bioelectronics enriches the toolbox of current biomedical technologies to promote translational biomedical innovations on smart wearables6, medical imaging, brain-inspired computing, and human-machine interfaces.
References
1 Zhang, S. & Cicoira, F. Flexible Self-powered Biosensors. Nature 561, 466-467, (2018).
2 Zhang, S. et al. Patterning of Stretchable Organic Electrochemical Transistors. Chemistry of Materials 29, 3126-3132, (2017).
3 Zhang, S. et al. Tuning the Electromechanical Properties of PEDOT:PSS Films for Stretchable Transistors And Pressure Sensors. Advanced Electronic Materials 5, 1900191, (2019).
4 Zhang, S. & Cicoira, F. Water-Enabled Healing of Conducting Polymer Films. Advanced Materials 29, 1703098, (2017).
5 Zhang, S. et al. Room-Temperature-Formed PEDOT:PSS Hydrogels Enable Injectable, Soft, and Healable Organic Bioelectronics. Advanced Materials 32, 1904752, (2020).
6 Zhang, S. et al. Hydrogel-Enabled Transfer-Printing of Conducting Polymer Films for Soft Organic Bioelectronics. Advanced Functional Materials 30, 1906016, (2020).

Solution-gated transistors have shown promising applications in biosensors due to the high sensitivity, low working voltage and the simple design of the devices. Solution-gated transistors normal have no gate dielectric and the gate voltages are applied directly on the solid/electrolyte interfaces or electric double layers near the channel and the gate, which lead to very low working voltages (about 1 V) of the transistors. On the other hand, the devices can be easily prepared by solution coating or printing methods because of the much simpler device structure compared with that of a conventional field effect transistor with several layers. Many biosensors can be developed based on the detection of potential changes across solid/electrolyte interfaces induced by electrochemical reactions or interactions. The devices normally can show high sensitivity due to the inherent amplification function of the transistors. In this talk, I will introduce several types of biosensors studied by our group recently, including DNA/RNA, glucose, dopamine, uric acid, cell, protein and bacteria sensors, based on flexible solution-gated organic thin film transistors. The biosensors show high sensitivity and selectivity when the devices are modified with functional nano-materials (e.g. graphene, Pt nanoparticles) and biomaterials (e.g. enzyme, antibody, DNA) on the gate electrodes or the channel. Furthermore, the devices are miniaturized successfully for the applications as sensing arrays. One important application is for the detection of SARS-CoV-2 IgG based on organic electrochemical transistors (OECTs). We have successfully developed an ultrafast, low cost, label-free and portable SARS-CoV-2 IgG detection platform using OECTs, which can be remotely controlled by a mobile phone. To enable a faster detection, voltage pulses are applied on the gate electrode of the OECT to accelerate the combination between the antibody and antigen. By optimizing the ion concentrations and pH values of test solutions, we realize specific detections of SARS-CoV-2 IgG within several minutes with a detectable region from 10 fM to 100 nM, which can cover SARS-CoV-2 IgG levels in human serum. It is expected that the solution-gated organic transistors will find more important applications especially wearable electronics for healthcare in the future.

Biocompatible conducting polymers (CPs) have been the subject of considerable interest in the decades following their discovery. As a class of material, CPs simultaneously possess several properties that make them uniquely suited for tissue engineering and neuroprosthetic applications. Compared to traditional metallic materials, CPs inhabit a unique niche of having multiple desirable properties all at once including mechanical flexibility, mixed ionic and electronic conductivity, good biocompatibility and a high degree of potential bio-functionalization. While CPs have been widely applied via electrodeposition on the surface of bioelectrodes, the effect of electrical parameters of the deposition was not comprehensively studied. Here, we systematically investigated and compared the effect of applied voltage, current and the time of deposition on physical and electrical properties. In addition, we evaluate the cellular response to those parameters.
We have investigated two common biocompatible CPs, poly(pyrrole) (PPy) and poly(3,4-ethylenedioxythiophene) (PEDOT), fabricated using galvanostatic (GSTAT) and potentiostatic (PSTAT) methods, and characterized the surface roughness, thickness, impedance, and charge storage density of the films. Electrodeposition was performed on microfabricated gold electrodes using a solution of 0.02M EDOT or 0.2M Py monomer and 0.2M poly(styrenesulfonate) dopant in DI water under GSTAT (0.1 to 1.5 mA/cm²) or PSTAT (0.5 to 0.95 V) deposition modes over intervals ranging from 1 to 10 minutes. Each surface was analyzed with materials confocal microscopy (MCM) to measure the CP film thickness and surface roughness. Additionally, the impedance and charge storage density (CSD) of the films were measured with impedance spectroscopy and cyclic voltammetry, respectively. Our results suggest a dual dependence of all measured properties (roughness, thickness, impedance, and CSD) on both deposition time and applied charge. For example, PPy films electrodeposited via GSTAT (0.1mA/cm² for 1 minute and 1.5mA/cm² for 10 minutes) on 50µm gold microelectrodes showed that the surface roughness (Rq) increased from 2.14±0.25nm to 12.0±0.84nm; thickness increased from 242.0±15.3nm to 2372.0±482.05nm . Comparing an uncoated gold microelectrode against a film deposited via GSTAT at 1.5mA/cm² for 10 minutes, CSD increased from 7.02mC/cm² to 98.7mC/cm²; and, impedance (at 100Hz) decreased from 2.38e6 Ω to 3.83e4 Ω. PPy properties exhibited generally linear trends, and PEDOT properties tended to display nonlinear trends. Additionally, PC-12 cells were cultured on these CP films to quantify changes in neurite outgrowth, cell morphology. Cells were cultured on substrates for 5 days, then fixed and stained for neurite outgrowth and nuclei. No statistically significant differences were observed between the experimental groups. These comprehensive results can be potentially utilized as a toolbox for the design of bioelectronics with desired physical and electrical properties.

30% of ‘in vitro’ blood pathogen cultures (BPC) yield false positives. US hospitals waste over $1M/y on these non-existent infections. The unreliability of BPC lead to large antibiotic and antiviral overuse, in turn creating more drug-resistant pathogens and more anxious testing. The CDC’s Antibiotic Resistance Threats in the US states that > 2.8 M/y of antibiotic-resistant infections occur in the U.S., leading to > 35,000 deaths/y.
In addition, BPC require large blood volumes (10-60 mL). When performed daily in hospitals, BPC lead to Hospital-Acquired Anemia (HAA) in 74% of patients and 100% of infants in the NICU.
BPC also require > 72 hrs for results as culture flasks can be incubated for up to 5 days depending on pathogens. Patients are prescribed antibiotics and antivirals pre-emptively well before the BPC results, leading to even more overuse and resistance.
The 4th issue for BPC are laboratory costs, which are out of reach for disaster areas, refugee camps, MD offices, and areas without health care infrastructure.
Increasing accuracy, speed, portability, and access while decreasing HAA, costs, and blood volumes in a reliable bioassay motivated this work to develop a new‘in vitro’ hand-held, portable blood diagnostic device called InnovaBug™ for rapid pathogen detection from 0.03 – 0.3 mL fluid drops. It can be used at the bedside, camps, or MD offices.
InnovaBug™ uses surface engineering and a new material, FluoBug™, to create super-hydrophilic coatings pre-applied to slides used as collection devices. A 2^nd key material is a cyanine dye, SYBR™ Safe Green, that can stain nucleic acid bonds specific to DNA and RNA, and reveal DNA and RNA strands.
InnovaBug™ is designed as a small handheld device using small fluid volumes. Its compact technology uses macroscopic epi-fluorescence of DNA/RNA over ~ 20 x 50 mm² areas instead of BCP or microscopy. Macroscopic epi-fluorescence can detect DNA/RNA via direct imaging in a biophysical assay on a 0.03-0.3 mL drop using dye/base pair ratios that are > 0.15 for DNA and > 0.015 for RNA.
This assay yields significant macroscopic epi-fluorescence within min. via small, inexpensive, calibrated excitations such as 395 or 365 nm LED ‘black’ lights. Next, a simple color analysis app, ‘Green Bug On Blue’ (GBOG™) quantifies pathogen detection and density in sub-mL drops via the relative ratio of green (520 nm) to blue (UVA 365-400 nm) light intensity in slide images captured on a smartphone camera. A small black box supporting the InnovaBug™ slides and the UV LED sources is closed to external light by aligning the smartphone camera on simple alignment guides on the black box top [2].
Proof-of-concept experiments tested InnovaBug™’s improvement for pathogen detection bioassays for blood, blood plasma, and urine tests. Yogurt water is used as a model system for infected blood and plasma because it can match their viscosity and optical properties as thin films and includes different bacterial species to identify and count. Analysis of epi-fluorescence across 6 sets of 6 InnovaBug™ prototypes shows that bacteria can be detected, identified, and counted using macroscopic epi-fluorescence.
Quantitative analysis via the ratio between green fluorescence intensity versus blue/UV excitation intensity by GBOG™ app is based on calibration curves obtained via State-of-the-Art Serial Dilution and Colony Forming Units (CFU) counting method. Comparing GBOG™ analysis and CFU counting shows pathogens are detected effectively and quantitatively via InnovaBug™ using DNA/RNA macroscopic epi-fluorescence. Integration of photo-electronics with microbiology achieved through InnovaBug™ has significant healthcare benefits for hospitals, disaster sites, MDs offices, and refugee camps.
[1] H. Zipper, et al. Nucleic Acids Res., 32 (12) (2004)
[2] Herbots N. et al, US & Internat. Pat Pending (2021)

Brain stimulation techniques are essential tools to address various neurological conditions. Approved neuromodulation techniques are either invasive in which patients have to undergo risky surgery or non-invasive but suffer from low spatial resolution. Focused ultrasound stimulation (FUS) is a promising new modality for non-invasive and minimally invasive brain stimulation which has been shown to elicit and suppress neural activity. In comparison to other non-invasive modalities, FUS offers comparable spatial resolution to invasive modalities. Stimulation is delivered deep into the brain using acoustic waves with sub-millimeter spatial resolution. However, the role of the FUS parameters is still heavily debated due to the limited performance of commercial FUS transducers and its inadequate pairing with typical in vivo neuronal recording technologies. To tackle the latter issue, Organ-on-chip (OoC), specifically brain-on-chip (BoC) is a promising new technology that can recapitulate human physiology using cerebral organoids in vitro. Mounting scientific literature evidences the superior physiological relevance of tissue response in OoCs compared to standard 2D static cell cultures. Multielectrode arrays are implemented on the BoC platform to record the neural activity of the organoids directly. We envision a tailored brain-on-chip platform could provide a suitable match for FUS to unravel its working principles, with the long-term goal of providing an electrical, thermal and mechanical sensing map of brain tissue response when locally stimulated by focused ultrasound. In this regard, one of the major technological drawbacks of ultrasound stimulation is the big form factor of the ultrasound transducers. Available ultrasound transducers are limited in terms of spatial resolution to study the effect of ultrasound in neuronal level and also bulky in terms of volume to be integrated into the context of BoC. We used microfabrication techniques to miniaturize the ultrasound transducer with the goal of having an ultrasound transducer with a small form factor with flexibility in the stimulation parameter. Lead zirconate titanate(PZT) piezoelectric film of various resonance frequencies is incorporated into an array on a silicon wafer. Unlike ultrasound transducer for imaging application, the acoustic pressure generated by the transducer is a crucial parameter of the transducer to induce neural activity, which can go up to 2 MPa for the central nervous system. In order to improve the spatial resolution of our transducer, a collimation technique based on slit diffraction is investigated to reach a single-neuron resolution. The resulting platform can be a step forward in understanding the mechanism of ultrasound neuromodulation by understanding the role of each ultrasound parameter and enabling the comparison of different neurons in an identical environment.

Significant advances in imaging technologies and molecular cellular biology have brought biological discoveries that boosted the understanding of biological phenomenon. Ranging from hematoxylin and eosin (H&E) staining to immunofluorescent staining (IF), different staining modalities have laid foundations for modern medicine, and next generation sequencing (NGS) (Metzker, 2009) caused a quantum leap in biological discoveries in a decade. While the imaging technologies provide information of the biological circuitry by providing spatial and structural information of a biological system, molecular cellular biology technologies provide the state of the biological cells in a more microscopic manner. However, to fully understand how the cells are functioning within spatial context, there needs to be a tool to connect the imaging technologies to molecular cellular biology. Such integrated technologies that can sort out cells with preserved spatial information will connect the data from spatial assays to that from the molecular assays.
Conventional cell sorting technologies, however, are mostly based on microfluidic cell sorting. Fluorescence activated cell sorter (FACS) is perhaps the most widely used cell sorter (Givan, 2013). FACS utilizes laser source to sort cells with the same fluorescence spectrum. These technologies require the cells to be dissociated into solution phase, losing the spatial information during the process. Intelligent image-activated cell sorting (Nitta et al., 2018) and Raman image activated cell sorting (Nitta et al., 2020) measured the cell image with machine learning and Raman image of the cells, respectively. These techniques are able to preserve the cellular phenotype information because the cells are sorted according to their images, but these cell sorting techniques still require dissociation of the cells and therefore the spatial context is lost before the cells are sorted. Spatially targeting cell isolating devices such as laser capture microdissection (LCM) (Espina et al., 2006) or similar methods are able to dissect out the regions of interest. Some platforms offer high resolution cell microdissection down to single cell level (Schütze and Lahr, 1998). However, the throughput for these platforms is three target retrievals per run (which takes approximately one hour), hindering their usage in cell sorting.
In this talk, we present a novel cell sorting methodology called spatially-resolved laser-activated cell sorting (SLACS) that preserves spatial information and demonstrate its usage in bridging spatial assay to various molecular assay.This is achieved by developing an innovative SLACS device which isolate cells on an indium tin oxide (ITO) coated slide glass into commercialized PCR tubes based on their image. The properties of cell sorting such as accuracy are demonstrated and the possibilities of post-processing such as DNA sequencing, RNA sequencing, or other molecular biology techniques are validated, proving the potential of SLACS for novel biological discovery tool by sorting mammalian cells to bacteria. Especially, we have reported a method of sequencing the genomes of small number of cells or single cell through this methodology (Kim et al., 2019, 2018).

Retinal prostheses are a promising option to provide artificial vision to individuals blinded by damaged photoreceptors in the retina. For the electrical stimulation of surviving retinal neurons, early commercialized retinal prosthetic devices have used intra-/extra-ocular cables for power/data transmission, however, these components introduce operation difficulties and/or increase the chance of post-operative complications. To address these issues, several studies have demonstrated the use of photovoltaics and photodiodes, including organic photovoltaic materials, which can generate electrical current without any external physical connections.
In the present work, we demonstrate the use of a high-efficiency, non-fullerene-acceptor (NFA) based organic photovoltaic layer (PCE10:ITIC:Y6) blend for retinal prosthetic applications. Among the three materials, PCE10 was selected because of its deep highest occupied molecular orbital (HOMO) which provides an improved electronic band structure compared to other organic materials (such as P3HT). Both ITIC and Y6 are recently developed NFAs which have been shown to provide superior photovoltaic performance with greater than 15% power conversion efficiency as well as broad spectral response and outstanding stability, making them excellent candidates for investigation as retinal prostheses.
We fabricated a retinal prosthesis devices using PCE10:ITIC:Y6/Au (100/20 nm) layers on ITO-coated glass substrates. The PCE10:ITIC:Y6 blend was spin-coated in an N₂-filled glove box. Then, Au was deposited as the top electrode using an e-beam evaporator. Lastly, polydimethylsiloxane (PDMS) was applied to encapsulate all parts but the center of the top electrode.
Photostimulation was conducted using a 450 nm LED (M450LP1, Thorlabs) which illuminated the light in the optical power density of 36.19 μW/mm² on the surface of the top electrode. The photocurrent of the fabricated device was evaluated in a recording chamber filled with Ames’ medium using patch pipettes in voltage-clamping mode. We located the patch electrode close (~10 μm) to the device surface, and then measured electrical artifacts resulted from the photovoltaic currents in response to 5-sec-long light stimuli. The photocurrents were indirectly calculated in comparison with electric artifacts measured in response to known current amplitudes delivered by a stimulus generator (STG2004, Multi-Channel Systems). As the light intensity increased from 20 to 100 % (36.19 μW/mm²), the peak photocurrent amplitude increased linearly from 53.58 to 242.94 nA/mm². Our previous study reported that electrical currents in similar amplitudes can evoke spiking activities in retinal ganglion cells (RGCs), suggesting that these photovoltaic devices can generate sufficient photocurrent for use as prosthetic retina.
To characterize the relationship between the stimulation duration and photocurrent, we illuminated devices for variable amounts of time ranging from 5 to 1 sec. As the irradiation time decreased, the photocurrent peak amplitude decreased exponentially from 242.94 to 110.94 nA/mm². We also tested the reproducibility of photocurrent response upon repetitive illumination. When devices were illuminated with 5-sec-long light pulses delivered at 0.1 Hz (20 repetitions), the photocurrent produced at the end of the sequence decreased by only ~10% compared to the initial response.
This study characterized the photo-response of a high-efficiency, NFA based organic photovoltaic layer (PCE10:ITIC:Y6) for retinal prosthetic use. Our results showed that fabricated devices were able to generate suitable photocurrent to stimulate RGCs; future experiments will investigate their spiking responses in RGCs. In addition to the wireless nature of the photovoltaic device, we expect the red-shift in stimulation wavelength of the blend results in less phototoxicity.
*Hyunsun Song and Hyeonhee Roh equally contributed to this work.

For the sensing of light, we have extremely powerful devices with the ability to detect even single photons. Similarly, the monitoring of force/pressure, e.g., for sound detection in acoustic communication is technically no problem. Only for chemical communication, for smell or taste detection on a technical level, we have (nearly) nothing. Despite the fact that monitoring chemicals in chemotaxis, i.e., the molecules-guided search for food of many organisms or the exchange of chemical cues between species as a way to communicate with each other, is the oldest of our sensory repertoire, we have essentially no technical device that offers the sensitivity and the bandwidth needed to sense and to differentiate many different odors. Earlier attempts to fill this gap by “artificial noses” failed (with the only notable exception being the “alcohol breath analyser” used by police) mostly because of lack of sufficient sensitivity.
In order to develop and present during this talk concepts for smell sensors that could overcome these sensitivity limits we will very briefly refer first to the world of smells and give a brief introduction into how mammalians and insects smell.
Using a biomimetic approach, i.e., using functional elements (proteins) from nature and combining them with electronic devices for hybrid transducers, we describe novel schemes and read-out concepts for smell sensors.

Traditional methodologies for bio-electrical interrogation are associated with interconnected leads and substrate-bound electrodes, which are mechanically invasive and lack intra-volumetric access [1]. Optogenetics, however, requires genetic modification, which limits its translational applications. Here we describe optically sensitive silicon nanowires (SiNWs) with p-i-n core-shell configuration that enable leadless, minimally invasive, and non-genetic photo-electrical modulation with unprecedented spatial resolution [2], [3]. Moreover, this approach allows us to perform bioelectrical interrogation of specific cells that are located within the 3D volume of viable tissues, such as the cardiac tissue. To demonstrate the utility of this approach, we have used this methodology to investigate bioelectrical coupling in cardiac and brain cells. To this end, we hybridized cardiac myofibroblasts with label-free silicon nanowires by spontaneous internalization. Live imaging of the hybridization process reviled that after internalization the cell-silicon hybrids were viable and active, and were able to undergo normal cell division in which the internalized SiNWs remained within one of the daughter cells. Then, we performed local stimulation with subcellular resolution by illuminating different nanowires that were internalized in a single cell. Consequently, transient calcium fluxes were induced, originating at the stimulation location demonstrating our ability to perform subcellular electrical interrogation. We then used these cell-silicon hybrids to study how myofibroblasts electrically couple to cardiomyocytes in vitro. Indeed, optical stimulation of the hybridized cells resulted in calcium propagation into neighboring cells, which demonstrated that myofibroblasts electrically coupled to other myofibroblasts and to cardiomyocytes in vitro, via gap junctions. We then performed statistical analysis of the propagation speed and coupling efficiency using tailored image analysis apparatus. The culmination of this study was to address the long-standing debate of whether myofibroblasts electrically couple with cardiomyocytes in vivo. When hybrids were injected into the left ventricular wall, they establish a seamless integration with the native heart in vivo, as opposed to bare silicon nanowires that resulted in a severe immune response, and fibrotic encapsulation. We then harvested the heart and applied local ex vivo cell-specific photo-stimulation of a specific pre-hybridized cell within the 3D tissue. Surpassingly, we found that hetero-cellular electrical coupling did not occur, as no calcium or electrical signal propagated from the stimulated myofibroblasts to the native tissue. Thus, we concluded that no electrical coupling between heterocellular myofibroblast-cardiomyocytes junction occurs in vivo as opposed to in vitro. To demonstrate the utility of this approach, we hybridized oligodendrocyte progenitor cells with SiNWs. The hybrids were then differentiated into oligodendrocytes and interacted with co-cultured neurons via myelin formation. Preliminary results demonstrate the ability of calcium transient to propagate from the hybrids to interfacing neurons, which suggests that they can be used for investigating the role of calcium transients in the myelination process. In summary, p-i-n SiNWs can be utilized for high spatial resolution optical modulation of cells for basic research investigations. It allows unprecedented spatial resolution in vitro, as well as within 3D viable tissues ex vivo.

Evaluation of sweet taste is essential for the survival of humans. Sweetness is the taste of carbohydrates such as monosaccharides and polysaccharides. The human taste system favors sweet carbohydrates, helping to ingest energy sources, but excessive sugar intake can cause various metabolic syndromes and can be life-threatening. Therefore, it is important to sensitively evaluate sweetness in various environments. There are various devices that can detect sweet taste substances, but there is no device that can simulate the human gustatory system in various environments. We developed an ultrasensitive bioelectronictongue that can mimic human sweetness based on the ligand binding domain of the human sweet receptor. We overexpressed only the ligand-binding domain (venus flytrap) of the human sweet taste receptor instead of the whole receptor. Then, the domain was immobilizedon the carbon nanotube field-effect transistor with floating electrodes. Our bioelectronic tongue can selectively detect sweet substances ata concentration of 0.1 fM, which is a 10^7-fold improvement in sensitivity compared to previous methods. This high sensitivity is due to the small size of the T1R2 VFT domain, which can be placed within the Debye length of the sensor surface. Furthermore, our sensors could show how our sweet taste receptors respond to sweet taste substances in a variety of environments. The sensor could evaluate sweeteners in real beverages such as apple juice and chamomile tea. In addition, we can evaluate the inhibition and enhancement of taste sensation by various chemical species such as zinc ions and amino acids. Importantly, our device showed significantly improved storability and reusability which could be important for future practical applications.

The development of electronic devices for neurosensing is leading to fundamental discoveries in communication setups for interfacing and computing the brain's electrical activity that is still a demanding task in the 21^st century. One of the greatest challenges for efficient neurosensing is to ensure that detection/transduction between biochemically rich systems and tools is fully mastered to reliably gather relevant information. In extracellular devices such as microelectrode arrays (MEAs), the discordance lies at the interface between ions and the electrodes. Engineering chemically/morphologically the electrode’s materials by decreasing its impedance, improving its affinity with neurons, and boosting its biocompatibility ensures better cell/electrode interface conditions to find the right materials that detect ionic signals from neurons and transduce them into electronic signals with the lowest information loss. Hence, the use of conducting polymers (PEDOT) has emerged for optimizing the performance of microelectrodes in neurosensing due to its mixed ionic electronic conduction, biocompatibility and low impedance. In parallel to the development of passive microelectrode, organic electrochemical transistors (OECTs) have received lots of attention in the biosensing field since they exhibit high coupling with cells and signal amplification. Notably, the transconductance represents an important parameter that depends on geometrical and material parameters that rules largely OECTs performances in biosensing.

In this direction, we explore the use of EDOT electropolymerization to tune post-fabrication material and geometrical parameters of passive microelectrodes for optimizing the cell/electrode interface by decreasing its impedance and improving its affinity with neurons (increasing the resistance “Rseal” that represents the cell/electrode cleft). For electropolymerized PEDOT MEAs, we demonstrate long term and stable extracellular recording of primary cortical neurons with a record signal-to-noise ratio (SNR) up to 37 dB (with ultra-low noise down to 2.1 μV RMS). Secondly, for active sensing with OECTs, this strategy exploits the concept of adaptive sensing where both transconductance and impedance are tuned simultaneously or independently. This approach shows an improvement of OECTs transconductance by 150%, volumetric capacitance by 300%, and a reduction in array’s variability by 60% in comparison with standard spin-coated OECTs.
The cytotoxicity of the electropolymerized EDOT was assessed for primary neural cells culture and no detrimental effect of electropolymerized EDOT on cell viability was observed. To extract the impedance and transconductance values for both MEAs and OECTs, we combine DC electrical measurements with electrochemical impedance spectroscopy (EIS). To show the cell/electrode morphology and neurite outgrowth to electropolymerized microelectrodes, Scanning Electron Microscopy (SEM) was performed. To correlate the morphological changes of the material with the enhancement of its electrical and electrochemical performances, Atomic Force Microscopy (AFM) in liquid and Raman Spectroscopy were achieved. Finally, in-vitro extracellular recorded signals from entorhinal cortex cultured slices and primary cortical neurons using both MEAs and OECTs are presented.
The key novelty of this technique is to propose a post-fabrication material engineering technique that can be used to optimize both passive (MEAs) and active (OECTs) devices for extracellular recording and promote new exploratory sensing strategies to ensure high quality neurosensing alternatives.

Health monitoring electric lenses include wireless biosensors that monitor biological signals in human diagnostics and wirelessly transfer the information to a receiver. In general, such wireless systems consist of in-parallel and/or in-series connection with a loop antenna inductor (L), a resistance (R), and a miniaturized ceramic capacitor (C) to resonate at a target frequency between transmitter/receiver circuits. However, conventional LCR resonator circuits make several problems such as low efficiency and short distance, so LCR-based wireless biosensors require the detection in high accuracy and practical use. Here we developed a prototype of a highly sensitive diagnostic contact lens with high sensitive biosensors and high efficient wireless transfer systems. [1-2]
[1]. Takamatsu, T.; Chen, Y.; Yoshimasu, T.; Nishizawa, M.; Miyake, T. Highly Efficient, Flexible Wireless Powered Circuit Printed on a Moist, Soft Contact Lens. Adv. Mater. Technol. 2019, 4.
[2]. Takamatsu, T.; Sijie, Y.; Shujie, F.; Xiaohan, L.; Miyake, T. Multifunctional High Power Sources for Smart Contact Lenses. Adv. Funct. Mater. 2020, 30, 1906225.

Ions and other biological carriers play an important role in controlling biological systems in biology. Bioelectronics has been established as a research discipline that focuses on the conversion of biological signals into electronic devices. We have been investigating the feasibility of transmitting H⁺ signals to biological systems to control the biological functions of enzymes^[1] and mitochondria^[2]. Here, we fabricated a biodevice that can transmit a proton signal from a device to a biological system via an external voltage, integrated sulfonated polyaniline (SPA) biotransducer on microelectrode, using ATP synthases for ATP synthesis/hydrolysis, and luciferase for bioluminescence fueled by ATP^[3]. When an input voltage is applied to the electrode, the SPA electrode can control the proton current and subsequent pH gradient modulation across the membrane, controlled the output bioluminescence via enzymatic cascade reactions from the ATP synthases and luciferase enzymes. This electron-ion transport platform provides a new way to control the ATP synthase reaction, and the proton signal is mediated by an external voltage.
[1] Miyake, T.; Josberger, E. E.; Keene, S.; Deng, Y.; Rolandi, M. An Enzyme Logic Bioprotonic Transducer. APL Materials 2015, 3 (1), 014906.
[2] Zhang, Z.; Kashiwagi, H.; Kimura, S.; Kong, S.; Ohta, Y.; Miyake, T. A Protonic Biotransducer Controlling Mitochondrial ATP Synthesis. Sci Rep 2018, 8 (1), 10423.
[3] Chen, Y.; Cui, M.; Lin, C.; Liu, B.; Mitome, N.; Miyake, T. Enzymatic Bioluminescence Modulation with an ATP Synthase Integrated Biotransducer. Adv. Mater. Technol. 2021, 2100729.