Symposium SB04—Bioelectricity in Microbial-Based Living Materials

Inherited retinal dystrophies and late-stage age-related macular degeneration, for which treatments remain limited, are among the most prevalent causes of legal blindness. Retinal prostheses have been developed to stimulate the inner retinal network; however, lack of sensitivity and resolution, and the need for wiring or external cameras, have limited their application. Here I report on the use of conjugated polymer nanoparticles (P3HT NPs), showing that they mediate light-evoked stimulation of retinal neurons and persistently rescue visual functions when subretinally injected in a rat model of retinitis pigmentosa. In particular I will report on recent results showing that the organic liquid prosthesis is effective also in retinas that have undergone a process of rewiring caused by the loss of photoreceptor function.

Cell membrane potential is known to be an essential parameter to monitor the electrical activities of excitable cells, such as neurons and cardiac cells. However, recent data has revealed that it also plays a crucial role in non-excitable cells, such as epithelial cells. Electrical perturbations induce localized bioelectrical changes, which can trigger different cell responses, including migration, mitosis, and mutation.¹
The directional migration of cells under an electric field is known as galvanotaxis, and represents a dominant mechanism in guiding the behavior of multiple cell populations in mammals, fishes, amphibians, and plants.^2,3 Wound healing is controlled by endogenous electric fields, generated by ion transport across the epithelium, which set a trans-epithelial potential difference (TCPD) of +40 mV in healthy conditions. At the wound, the TCPD falls to zero, but it is maintained at normal levels in its proximity (~ 0.5 mm) to drive epithelial cells to migrate and heal the wound.⁴ Bioelectrical cues are also involved in cellular mutation, responsible for the onset of multiple diseases. For example, cell electrical charges are the biophysical manifestation of metabolic patterns in cancer development and metastasis.⁵ Cancer cells exhibit the Warburg Effect, resulting in the secretion of lactate anions that produce a high concentration of negative surface charges.⁵ The membrane potential is also highly correlated with mitosis, DNA synthesis, cell cycle progression and overall cellular proliferation.⁶
Understanding these mechanisms is the key to unravel precision diagnostic and therapeutic strategies. However, this dynamic information is currently hard to access because existing technologies for electrophysiological recording of resting cell membrane potentials are invasive: they require physical access to the intracellular compartments, which implies the disruption of the cell membrane (known as electroporation).⁷ This makes it challenging to perform passive measurements and prolonged recordings under varying conditions.
A variety of alternative or complementary technologies based on optical methods were introduced in the past decades. However, the majority of them are designed to be implemented in electrogenic cells, which transport signals through action potentials (i.e., rapid polarization and depolarization of the cell membrane). Nevertheless, most of the cells in the human body do not fire action potentials, and their mechanisms of communication happen in a narrower signal range.
In this work, we present a novel approach in electrophysiological recording, based on optical mirroring of bioelectric charges. The feasibility of this method was previously demonstrated for the high throughput recording of action potentials in human-derived cardiomyocytes.⁷ In this context, the technology was refined and implemented in epithelial cells.
The nano-platform consisted of two chambers separated by a thin membrane. Pass-through electrodes enabled to mirror the cell membrane charge (top) all the way to the optical chamber (bottom), where dispersed fluorophores migrated to balance the charge, generating a dynamic fluorescent signal. Fluorescence was recorded with a camera, while exciting the fluorophores with incident light. Preliminary results on HEK cells yielded a fluorescence intensity increase of the 2.5% in presence of a cell on the microelectrode compared to reference microelectrodes. At present, this technology may be used to evaluate cell-substrate adhesion, monitor cell proliferation, and test novel biomaterials. In the near future, further investigations could allow extrapolating quantitative data on resting membrane potential in dynamic settings to perform cell/tissue studies on wound healing, cellular division, and cancer progression.

References:
¹ Huang, Y. J. et al. Sci Rep 2016, 6, 21583.
² Zhao, M. et al. Nature 2006, 442 (7101), 457-60.
³ McCaig, C. D. et al. Physiol Rev 2005, 85 (3), 943-78.
⁴ Song, B. et al. Proc Natl Acad Sci U S A 2002, 99 (21), 13577-82.
⁵ Vieira, A. C. et al. PLoS One 2011, 6 (2), e17411.
⁶ Kadir, L. et al. Front Physiol 2018, 9, 1661.
⁷ Barbaglia, A. et al. Adv Mater 2021, 33 (7), e2004234

Light-driven modulation of cellular activity with both high spatial and temporal resolution is becoming an increasingly active research area. Existing approaches, such as optogenetics, have shown promising results and, as an alternative, we envision the use of light-sensitive molecules that act as photo-actuators avoiding genetic manipulation.
We are interested in investigating the effects of different photo-actuators, such as azobenzene-based photochromic and donor-acceptor molecules. These compounds are designed to satisfy the following requirements: (i) absorb light in the visible or near-infrared window of the spectrum; (ii) spontaneously and efficiently partition into the lipid bilayers owing to their amphiphilicity, (iii) exhibit low toxicity in dark condition and preferentially also under illumination.
We combine the observation of the photoinduced effects on both HEK-293 cells and primary neurons, which are evaluated by exploiting electrophysiology, while steady-state and time-resolved optical spectroscopies reveal their photophysics and functioning mechanism.
Taken together, our data allows us to identify different light-driven mechanisms, responsible for the photoinduced effects observed on cells. These include, for instance, (i) the thinning or thickening of the membrane due to conformational changes in the molecules, (ii) the increase of the membrane permeabilization, and the formation of pore-like structures likely due to the lipid peroxidation following the photosensitization of singlet oxygen within the cell membrane, (iii) the rearrangement of the charges adsorbed to the membrane due to variations in the molecular dipole moment.

Establishing close interactions between biological systems and synthetic materials is the key to forming biohybrid assemblies that find use in sensors, actuators, and robotics. In this work, I will present an electronic platform based on an n-type conjugated polymer that is tailored to form favorable interactions with catalytic enzymes.¹ When this biohybrid is applied in an enhancement mode organic electrochemical transistor (OECT), the device detects glucose and lactate in blood serum or saliva with excellent sensitivity and selectivity over six orders of magnitude wide detection range. The same biohybrid serves as the anode of a glucose fuel cell, extracting enough power from bodily fluids to drive the microscale OECT sensors. While showing the unique characteristics of these devices, I will discuss the possible pathways through which the polymer film generates charges as the enzyme reacts with its metabolites.² I will then show how these biohybrids are also responding to visible and NIR light by generating photocurrents in aqueous electrolytes.
1 Ohayon et al Nat. Mater., 2020, 19, 456.
2 Druet et al Adv. Electron. Mater. 2022, 2200065.

Challenged by a changing climate, dwindling natural resources, and a growing global population, we need advanced renewable materials that meld the sustainability of biological materials with the functionality of conventional materials. To help address this need, my research group creates sustainable and environmentally-responsive living materials by seamlessly integrating conventional materials with living systems. One major effort in our group is to co-engineer microorganisms and microelectronics to serve as living bioelectronic sensors that can selectively sense a variety of complex molecules in real-time.

In my talk, I will first describe a collaborative project with the Silberg group at Rice to develop bioelectronic sensors that have minute detection times. We programmed Escherichia coli to produce current in response to specific chemicals using a modular, eight-component, synthetic electron transport chain. As designed, this strain produced electrical current upon exposure to thiosulfate, an anion that causes microbial blooms, in two minutes. By incorporating a protein switch into the synthetic pathway and encapsulating the bacteria with conductive nanomaterials, this amperometric sensor could be modified to detect an endocrine disruptor within two minutes in riverine water. Our results provide design rules to sense a variety of chemicals with mass transport-limited detection times and a new platform for miniature, low-power bioelectronic sensors that safeguard ecological and human health.

In the second part of my talk, I will describe how we have created bioelectronic sensors that convey multiple channels of information. Existing bioelectronic sensors can sense a variety of hazards to human and environmental health, however, these sensors transmit information through only a single electrochemical channel. This severely limits the amount of sensing information that can be transmitted. To increase this information content, we developed a multichannel bioelectronic sensor in which different chemicals modulate distinct extracellular electron transfer pathways in E. coli. To create an E. coli strain with two reporting channels, we introduced a riboflavin synthesis pathway from Bacillus subtilis alongside the metal reducing (Mtr) pathway from Shewanella oneidensis. We can distinguish whether one or both pathways are active in this strain using either chronoamperometry or a series of amperometric measurements at distinct redox potentials. To demonstrate multi-channel bioelectronic sensing, we regulated the Mtr and riboflavin pathways using arsenic and cadmium responsive promoters. With this strain, we used a series of amperometric measurements at distinct redox potentials to distinguish the presence of the different heavy metals in situ. These accomplishments provide a new platform for multichannel bioelectronic sensors that simultaneously detect and report multiple toxins.

Diatoms are early Jurassic microalgae, a photosynthetic group with a major environmental role on the planet due to the biogeochemical cycling of silica and global fixation of carbon. However, they can evolve into harmful blooms through a resourceful communication mechanism, not yet fully understood. Harmful algae blooms in water supply reservoirs must be eradicated due to unwanted toxin production and filter blocking at water treatment works. Until now there has been no efficient consensus that harmful microorganisms such as diatoms communicate with each other and no accurate and self-sustained tool to monitor such communication.

Here, we demonstrate the ability to electrically monitor a diatom cohort. The breakthrough is realized by means of a unique measurement setup based on low impedance electrodes that exploit the large Helmholtz-Gouy-Chapman double-layer capacitance. The sensing system comprises a transducer based on a metal/Si/SiO₂/Au electrode. Small extracellular voltages of diatoms adhered to the electrode induce a displacement current that is enhanced by a gain factor proportional to the double-layer capacitance.

The origin is paracrine signalling, which is a feedback, or survival, mechanism that counteracts changes in the physicochemical environment. The intracellular messenger could be related to Ca²⁺ ions since spatiotemporal changes in their concentration match the characteristics of the intercellular waves. Our conclusion is supported by using a Ca²⁺ channel inhibitor. The transport of Ca²⁺ ions through the membrane to the extracellular medium is blocked and the intercellular waves disappear. The translation of microalgae cooperative signalling paves the way for early detection and prevention of harmful blooms and an extensive range of stress-induced alterations in the aquatic ecosystem.

Over the last two decades, there has been a wealth of research in using microbially modified electrodes for various applications from microbial fuel cells to biosensing to electrosynthesis. The majority of these technologies are limited by slow extracellular electron transfer between the microbe and the electrode material. This talk will discuss chemical, materials, and biological strategies for improving extracellular electron transfer. It will start with a discussion of materials modification strategies for improving the surface area and biotic-abiotic interface to enhance current densities followed by a discussion of chemical strategies for mediating electron transfer. Finally, the use of synthetic biology to genetically engineer microbes to communicate directly with electrode surfaces will be discussed and the talk will compare and contrast the chemical, materials, and biological strategies for improving electron transfer and therefore performance of these bioelectrochemical systems.

The keywords solar power and electricity combined evidently point our minds toward photovoltaics. However, plant microbial fuel cells (PMFCs) have a similar working principle. The technology – which emerged in 2008 – combines living plants and microorganisms in an in situ fuel cell configuration, also converting sunlight into electricity [1]. But how do these two technologies compare? In this study, PMFCs were approached from a photovoltaic perspective. A rough upper limit for the overall energy conversion efficiency was estimated and compared to various classes of photovoltaics. To this end, each intermediate step in the working principle of plant microbial fuel cells was investigated, and of each step, the maximum efficiency found in literature was used to calculate the maximum power conversion efficiency. This approach estimated the maximum efficiency to be around 1%, more than one order of magnitude lower than traditional and emerging photovoltaics [2].
Going from theory to practice, the corresponding maximum power density was compared to the historical evolution of reported power output values. However, no apparent increase through time could be seen since their emergence. The application of the technology may explain this as the plant microbial fuel cell has many dedicated niche applications ranging from sensing to remediation, where power output might not be of interest [3]. Even though various routes toward increased power output are still being proposed, it is clear that PMFCs are intrinsically limited and dwarfed in comparison to photovoltaics when looking at power output alone. Nonetheless, the various niche applications - together with certain advantages the technology has over photovoltaics, i.e., aesthetics, price, straightforwardness, etc. - provide an added value to the technology.
This critical discussion aids in placing the plant microbial fuel cells, their applications, and expectations in the world of green, and particularly of solar energy-related research.

[1] Strik D.P.B.T.B. et al., Int. J. Energy Res. 32 (2008)
[2] NREL - Best Research-Cell Efficiency Chart (2022)
[3] Ebrahimi A. et al., J. Environ. Chem. Eng. 9 (2021)

The bio-sensitized solar cell is the architecture with the highest power conversion efficiencies (PCE) amongst photovoltaic devices that integrate natural photosynthetic proteins (1-2%). In these devices, a nanostructured wide-bandgap semiconducting electrode is coated with the protein and used as the photoanode in an electrochemical cell. Our team studies two different biosensitizers that stand out as some of the best performing natural light absorbers in bio-photovoltaic devices: bacteriorhodopsin (bR), a light-driven proton pump from archaebacteria, and photosystem I (PSI), a chlorophyll-protein complex from cyanobacteria and green plants that catalyzes photoactivated unidirectional electron transfer in oxygenic photosynthesis. Two factors that currently limit the charge transfer efficiency from the protein to the TiO₂ are the reduced contact area between the two materials and the non-specific orientation of the protein with respect to TiO₂. The proteins here studied are significantly larger than traditional synthetic dyes, which hinders their penetration into the pores of the TiO₂ nanoparticle layer during impregnation. The use of TiO₂ nanorods doped with CNTs is proposed to provide larger contact area where the protein can inject the photoelectrons into the semiconductor. In nature, electron transfer occurs in very specific electrical pathways that have evolved over millions of years, while simple dropcasting of a protein onto the electrode results in random orientations that may or may not connect these natural electrical wires with the electrode surface. Alternative techniques to promote an organized interface with preferential orientation of the proteins on the TiO₂ are here explored, so that the electron injection is directional according to the natural function of the biomolecule in the organism. In situ crystallization and structural modifications of the protein are proposed to drive a preferential orientation on the electrode. The effect of these variables in photovoltaic performance of devices is determined. The devices here proposed consist on the biosensitized TiO₂ photoanode and a PEDOT/CNT counterelectrode. A gel electrolyte is used to connect the two electrodes, containing the hydroquinone/benzoquinone pair or aqueous-soluble bipyridine cobalt(II/III) complexes as direct redox mediators for bR and PSI, respectively. These work intends to deepen the understading of electron transfer in a bio-hybrid interface for photogeneration of bioelectricity.

Biological systems are notoriously difficult to control. A major technical issue is controlling where and when bio-adhesion occurs on surfaces in engineered systems. Promoting bio-adhesion may be useful to pattern cells for tissue engineering. Preventing bio-adhesion is useful in micro-algal photobioreactors, where cleaning operations are costly and time consuming.

Current methods to promote or inhibit bio-adhesion depend on directly modifying the surface chemistry (e.g. hydrophobicity, molecular end groups). Here, we study the interaction between micro-algae cells (i.e. freshwater Chlorella vulgaris and saltwater Nannochloropsis oculata), and an engineered surface that can actively change its zeta potential based on the magnitude and polarity of applied voltage without changing its chemistry. Since the studied algae have negative zeta potentials, colloidal DLVO theory suggests that a surface with negative zeta potential should repel algae and a surface with positive zeta potential should attract algae. We observe that algae adhesion is inhibited at negative zeta potentials and enhanced at positive zeta potentials with a power draw of ~nW by applying -/+ 1V. Using microfluidic experiments, we report the effect of varying voltage magnitude, voltage polarity, wall shear, algae species, and solution salinity on bio-adhesion. We also conduct longer term (4 week) bio-adhesion and patterning experiments to observe the effect on practical performance

Bioelectronic interfaces significantly affect the signal transmission between electronic devices and biomaterials. Therefore, the electrical characterization of bioelectronics interfaces is of paramount importance for designing various bioelectronic devices or biohybrid sensors. Conventionally, the characterization of bioelectronic interface impedance relies on estimation using electrochemical impedance spectroscopy (EIS) data. However, the conventional method possesses a critical limitation in that the results of the electrical characterization are not unique since different estimations can produce identical impedance results.¹ The limitation undermines the reliability of the measurements and can causes errors. Thus, the development of a method that can accurately distinguish and measure the impedance of the bioelectronic interface and bulk is necessary.
In this study, we propose a novel method that satisfies the necessity by applying the transfer length method in an electrode/electrolyte system. Transfer length method-based interface electrical characterization (TLM-IEC) obtains accurate bulk electrolyte impedance by measuring impedance between electrodes at various intervals, and then the interface impedance is obtained by removing the bulk electrolyte impedance from the total impedance. The validity of the TLM-IEC was confirmed, and the method exhibits a superior advantage over the conventional method since the impedances of the interface and bulk are measured, not estimated. The unique electrical characterization of bioelectronic interface by the TLM-IEC can facilitate the optimized design of bioelectronic devices and sensors, and can be utilized for various applications.

Reference
1. Y. Liu, Y. Bai, W. Jaegermann, R. Hausbrand, and B. X. Xu, "Impedance Modeling of Solid-State Electrolytes: Influence of the Contacted Space Charge Layer," ACS Appl. Mater. Interfaces, vol. 13, no. 4, pp. 5895-5906, Feb 3 2021.

Cells generate a -10 mV to -100 mV electrical potential across the plasma membrane driven by an ion gradient. This resting membrane potential, in comparison to the action potentials of neurons and muscle cells, is present in all cells, including bacteria. A fundamental understanding of bacterial electrophysiology would lead to new methods for the control of engineered living materials, as well as significant advances in synthetic biology, cell growth in industrial bioreactors, and screening of anti-bacterial agents. Recent research in the Payne Lab is aimed at understanding bioelectricity in bacteria guided by questions of heterogeneity and cell growth with applications in materials science.The resting membrane potential of individual cells within a population is highly heterogeneous. In addition to cell-level heterogeneity, we also observe temporal heterogeneity with 2% of individual cells showing short bursts of hyperpolarization, referred to as “spiking.” We have developed methods to image and control resting membrane potential, simultaneously, on the single cell level to determine the underlying source of both the cellular and temporal heterogeneity. We use blue light, which can be patterned, to suppress cell growth in certain regions, creating patterned bacteria, in a way that is not possible using diffusible reagents that affect all cells equally. Blue light-mediated hyperpolarization is coupled with a range of cellular responses including generation of reactive oxygen species, altered enzymatic activity, and changes in membrane permeability, in addition to the inherent heterogeneity of this system. Our experiments are aimed at decoupling these cellular responses to determine what factors drive the associated decrease in cell growth. We hope these experiments will provide new insights in the growing field of bacterial electrophysiology.

Water sources in both gaseous and liquid forms are ubiquitous. The electrostatic and hydrokinetic energy associated with this water is a large reservoir that potentially could be transformed into a sustainable source of energy for powering electronics. We have found that useful amounts of electricity can be generated from water with microbial materials. For example, protein nanowires harvested from the microbe Geobacter sulfurreducens are the functional material in ‘Air-gen’ devices that continuously generate electricity from the ambient humidity that is available literally anywhere on earth. We also found that devices fabricated from microbial biofilms can generate electricity from evaporating liquid water, achieving energy densities higher than those possible with traditionally engineered materials. These two types of devices for generating electricity from water are complementary, closing the loop for broadly harvesting electricity from all distributed water forms on earth. We further demonstrate the practical application of both types of devices for continuous, sustainable powering of wearable electronic devices that monitor important human physiological functions.

The study of human-machine interaction is an appealing research area where the developed interfaces find application in robotic,^1,2 biological^3,4 and medical areas.⁵ In particular, great interest is rising for the development of a contactless and wireless interaction. Within this context, light represents a clean and spatiotemporal precise tool to achieve such a goal.⁶ Conjugated molecules and polymers offer a promising platform to interface with cells and living organisms due to their high optical absorption/emission cross section, chemical synthesis’ easiness and relatively low toxicity.^7,8
In this work, we focus on cardiomyocytes photopacing, whichare interesting candidates to test the stimulation process due to their excitability, contraction ability and their electrical behaviour. Specifically, we present the use of an azobenzene-derivative that is able to trigger the signalling process in muscle cells at all the physiological levels of the contraction. We characterized the effect of the photostimulation on electrophysiology response, calcium wave generation and contraction activity. Moreover, we test this approach on in vitro micro-physiological systems controlling its macroscopic contraction activity.
This last step consists of the realization of a bio-hybrid photoresponsive smart tissue, which is crucial for moving towards a real translational application and achieve the ultimate goal of this project. This can be a breakthrough for future applications in robotics and pharmacology, as well as tissue regeneration.

References
1. Feinberg, A. W. Biological Soft Robotics. Annu. Rev. Biomed. Eng. 17, 243–265 (2015).
2. Ricotti, L. et al. Biohybrid actuators for robotics: A review of devices actuated by living cells. Sci. Robot. 2, eaaq0495 (2017).
3. Antognazza, M. R., Abdel Aziz, I. & Lodola, F. Use of Exogenous and Endogenous Photomediators as Efficient ROS Modulation Tools: Results and Perspectives for Therapeutic Purposes. Oxid. Med. Cell. Longev. 2019, 1–14 (2019).
4. Colombo, E., Feyen, P., Antognazza, M. R., Lanzani, G. & Benfenati, F. Nanoparticles: A Challenging Vehicle for Neural Stimulation. Front. Neurosci. 10, (2016).
5. Maya-Vetencourt, J. F. et al. A fully organic retinal prosthesis restores vision in a rat model of degenerative blindness. Nat. Mater. 16, 681–689 (2017).
6. Vurro, V., Venturino, I. & Lanzani, G. A perspective on the use of light as a driving element for bio-hybrid actuation. Appl. Phys. Lett. 120, 080502 (2022).
7. Di Maria, F., Lodola, F., Zucchetti, E., Benfenati, F. & Lanzani, G. The evolution of artificial light actuators in living systems: from planar to nanostructured interfaces. Chem. Soc. Rev. 47, 4757–4780 (2018).
8. Hopkins, J. et al. Photoactive Organic Substrates for Cell Stimulation: Progress and Perspectives. Adv. Mater. Technol. 4, 1800744 (2019).

Integration of synthetic electronic biomaterials with living systems has emerged as an approach for modulating or probing cellular behavior and tissue function that minimize the need for contact with traditional, bulky, and non-flexible inorganic electrodes. Bioelectronic materials have been demonstrated to effectively integrate with cells, in vitro tissue models, organoids, and even with small model organisms, but the long-term effects of these materials on living systems are rarely considered when designing a material. In this presentation, we present peptide-based structures functionalized with π-conjugated units as designer biomolecules that can be interfaced with excitable cells. We present herein structure-function relationships that demonstrate the dependence of assembly behavior on monomeric structure and assembly trigger used while in completely aqueous environments. First, we show that small molecule coupling agents that allow for anhydride formation generate peptidic π-conjugated conduits that are amenable to disassembly once the fuel is diminished and anhydride hydrolysis predominates. The compliance of a peptide-bearing quaterthiophene monomer to this process is highly dependent on the electrostatic nature of the peptide sequence used. On the other hand, peptide-polydiacetylene conjugates will also be discussed as π-conjugated assemblies that exhibit sequence-dependent photophysical and electrical properties. Our results show the differential impacts of amino acid size and polarity, and the degree that these molecular parameters can influence bulk properties based on the location of the residue substitution. Interestingly, we also show that the interaction of fibroblasts (as a model cell line) with the monomeric and polymeric equivalents of these peptide-polymer also sequence-dependence. Collectively, these findings shed light to the tunability of properties and assembly behavior of π-conjugated peptides under physiologically relevant environments. In the future, we will leverage these design principles for engineering biotic-abiotic interfaces that rely on the properties of these peptides functionalized with π-conjugated units.

The ability of certain materials to regenerate after damage has attracted a great deal of attention since the ancient times. For instance, self-healing concretes, able to resist earthquakes, aging, weather, and seawater have been known since the times of ancient Rome and are still the object of research.
While the field of mechanically healable materials is relatively established, self-healing conductors are still rare, and are nowadays attracting enormous interest for applications in electronic skin for health monitoring, wearable and stretchable sensors, actuators, transistors, energy harvesting, and storage devices, such as batteries and supercapacitors. Self-healing can significantly enhance the lifetime of conducting materials, leading to the improved environmental sustainability and reduced costs.
Conducting polymers exhibit attractive properties, such as mixed ionic-electronic conductivity, leading to low interfacial impedance, tunability by chemical synthesis, ease of process via solution process and printing, and biomechanical compatibility with living tissues, which makes them ideal materials for bioelectronics and stretchable electronics. However, they show typically poor mechanical properties and are therefore not suitable as self-healing materials. Self-healing conductors can be achieved upon mixing with other polymers, such as poly(vinyl alcohol) (PVA) and poly(ethylene glycol) (PEG), which provide the mechanical characteristics leading to self-healing.
My talk will deal with processing and characterization of conducting polymer films and hydrogels and devices for healable, flexible, stretchable and electronics as well as for implantable electrodes. I will particularly focus on processing strategies to fabricate stretchable and self-healing conductors and their applications. [1-9]
References
Y. Li, X. Zhou, B. Sarkar, N. Gagnon-Lafrenais, and F. Cicoira, Adv. Mater. 2108932, 2022.
Y. Li, X. Li, S. Zhang, L. Liu, N. Hamad, S. R. Bobbara, D. Pasini, F. Cicoira, Adv. Funct. Mater., 30, 2002853, 2020.
Y. Li, X. Li, R. N. Unnava Venkata, S. Zhang, F. Cicoira, Flexible and Printed Electronics 4, 044004, 2019.
N. Rossetti, P. Luthra, J. Hagler, A. H. J. Lee, C. Bodart, X. Li, G. Ducharme et al., ACS Appl. Bio Mater. 2, 5154-5163, 2019.
[5] C. Bodart, N. Rossetti, S. Schougaard, F. Amzica, F. Cicoira et al., ACS Appl. Mater. Interfaces, 11, 17226-17233, 2019.
[6] S. Zhang, Y. Li, G. Tomasello, M. Anthonisen, X. Li, M. Mazzeo, A. Genco, P. Grutter, F. Cicoira, Adv. Electron. Mater, 1900191, 2019.
S. Zhang, F. Cicoira, Adv. Mater. 29, 1703098, 2017.
X. Zhou, A. Rajeev, A. Subramanian, Y. Li, N. Rossetti, G. Natale, G. A. Lodygensky, and F. Cicoira, Acta Biomaterialia, 139, 296-306, 2022.

Photosystems II and I (PSII and PSI) are the reaction centre complexes that drive the light reactions of photosynthesis. PSII performs light-driven water oxidation (quantum efficiencies and catalysis rates of up to 80% and 1000 e−s−1, respectively) and PSI further photo-energises the harvested electrons (quantum efficiencies of ~100%). The impressive performance of the light harvesting components of photosynthesis has motivated extensive biological, artificial and biohybrid approaches to re-wire photosynthesis to enable higher efficiencies and new reaction pathways, such as H2 evolution or alternative CO2 fixation. To date these approaches have focussed on charge extraction at the terminal electron quinones of PSII and terminal iron-sulfur clusters of PSI. Ideally electron extraction would be possible immediately from the photoexcited reaction centres to enable the greatest thermodynamic gains. However, this was believed to be impossible because the reaction centres are buried around 4 nm within PSII and 5 nm within PSI from the cytoplasmic face. Here, we demonstrate using in vivo ultrafast transient absorption (TA) spectroscopy that it is possible to extract electrons directly from photoexcited PSI and PSII, using both live cyanobacterial cells and isolated photosystems, with the exogenous electron mediator 2,6-dichloro1,4-benzoquinone (DCBQ). We postulate that DCBQ can oxidise peripheral chlorophyll pigments participating in highly delocalised charge transfer (CT) states after initial photoexcitation. Our results open new avenues to study and re-wire photosynthesis for bioenergy and semi-artificial photosynthesis.

Engineered living materials have many exciting properties such as self-healing, bioresponstivity, and the formation of composite materials with complex structures. One challenge in the advancement of engineered living materials is controlling the growth of single cells to form micro- and nano-scale patterns. We, and others, have reported the use of low intensity blue light to control the resting membrane potential of bacteria at the single cell level. We found that blue light (480 nm, <60 s) can slow the growth of B. subtilis and E. coli without damaging the cells. The growth rate of bacteria is inversely proportional to blue light exposure time. We have used this method to pattern bacteria by controlled cell growth. This use of blue light provides a low-cost, high-throughput method for patterning biofilms or the deposition of materials by bacteria, such as bacterial cellulose and calcium carbonate. Current research is aimed at characterizing the underlying mechanism for the slowed bacteria growth including hyperpolarization, generation of reactive oxygen species, and enzyme activity.

In the natural photosynthesis of plant or cyanobacteria, functional proteins and redox cofactors are sophisticatedly self-assembled in the thylakoid membrane, exhibiting nearly 100 % of light-harvesting quantum efficiency. Photosystem I (PSI) is an integrated membrane protein complex that catalyzes the transfer of electrons using light energy. The photon energy absorbed by PSI also provides a proton motive force used to produce organic compounds. This finding has inspired engineers to pursue biomimetic and bio-inspired designs for efficient light-harvesting and charge extraction. However, the efficiency of nano-bio hybrid photosystems is often limited by the poorly-oriented assembly of photosystems with nanomaterials. In this work, we assembled PSI with protonated-carbon nitride (PCN) nanosheets through balanced intermolecular interactions, resulting in linker-free PSI/PCN hybrid nanostructures that generate favorable electronic interfacial structures for charge transfer through a Z-scheme system. Surface-modified carbon nitrides can provide photocatalytic activity under visible light irradiation conditions and a new electron transfer path in the hybrid structure with PSI. Electrostatic hybridization between protonated sites on the PCN nanosheets and the partially negatively charged lumen side of PSs provides a favorable orientation for efficient electron transfer. The PSI/PCN hybrids exhibit a photocurrent density more than 28 times higher than randomly oriented PSI. We also show that when coupling the developed bio-photocathode with a PSII-based photoanode, a photocurrent of 7.7 μA cm^-2 under bias-free conditions and a cell power up to 0.9 μW cm⁻² can be achieved. This study provides a vital strategy to construct an efficient biochemical photoelectrode based on natural photosystems coupled with semiconductor carbon nanostructures through simple self-assembly in a favorable orientation for efficient and stable interfacial electron transfer. This work was supported by Nano●Material Technology Development Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (NRF-2017M3A7B4052797).

At least 50% of the wastewater from human activities is discharged into the environment without treatment, a practice that compromises the human right to access clean water and sanitation. Microbial fuel cells (MFCs) are devices capable of converting chemical energy into electrical energy through electrochemical reactions, using the electrosynthesis of microorganisms. In this work, a device composed of two chambers is proposed. Biodigester effluent is used as an anolyte and oxidation of organic components is perfomed by bacteria at the electrode surface in the anodic chamber, while the reduction of heavy metals from industrial wastewater takes place in the cathodic chamber. The proposed MFC can simultaneously reduce the load of pollutants in agricultural and metallurgic wastewater and produce renewable energy. The objective of this study is to determine the effect of coating the graphite electrodes in the cathodic and anodic chamber with carbon nanomaterials, including graphene and a seamless hybrid of graphene and carbon nanotubes, both materials obtained by chemical vapour deposition. The nanostructured electrodes provide a larger surface area and increased electron transfer rates to improve the performance of bioelectricity generation. This work improves the comprehension of bioelectrochemical phenomena occurring in biofilm-based microbial devices and analyzes how the electrode surface structure can tune the charge transfer efficiency at the interface of living organism and carbon electrodes.

Flexible, soft, self-healing, and adhesive conductive materials with Young’s modulus matching biological tissues (with a moduli of 0.5-500 KPa) are highly desired for applications in bioelectronics. Here, we report self-healing, stretchable, highly adhesive, and conductive hydrogels obtained by mixing polyvinyl alcohol (PVA), borax, and Poly (3,4-ethylenedioxythiophene) doped with polystyrene sulfonate (PEDOT:PSS) screen printing paste. The prepared hydrogels demonstrated remarkable compliance on skin, outstanding plastic stretchability (over 10000% strain), high adhesion on pig skin (1.96 N/cm²), a good strain sensitivity (gauge factor = 3.88 at 500 % strain), remarkable self-healing properties, and low compressive Young’s modulus (~3.7 KPa). Our hydrogels were used to fabricate electrophysiological epidermal patch electrodes, which exhibited high-quality similar to commercial Ag/AgCl gel electrodes for recording of electrocardiography (ECG) and electromyography (EMG) signals.

Monitoring the vital signs of children born prematurely is of paramount importance for their health. Electrocardiography (ECG), electromyography (EMG), heart rate and respiration data are used to assess their well-being and development. These measurements require the placement of electrodes on the skin via an adhesive, which can lead to damage during its removal because the skin of premature infants does not have a protective outer layer. Therefore, alternative monitoring methods are needed to increase comfort and reduce the burden of preterm infants. This can be achieved with flexible, stretchable, and self-adhesive electrodes that can adapt to the shape and movements of the child while maintaining signal quality for a long-term use.
Commonly used biomedical electrodes for on-skin electrophysiological measurements are hydrogel-based and suffer from water loss, therefore degrading and losing signal quality overtime. Long-term monitoring using gels is therefore hindered. On the other hand, electrodes using adhesives often adhere too strongly for sensitive skin and can only be used once. Herein, a conductive polymer, poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS), is mixed with a short chain polyurethane diol (PUD) and printed onto a thin thermoplastic polyurethane substrate to obtain a reusable, wearable and dry electrode for durable electrophysiological signal monitoring. Due to its light adhesivity, the dry electrode can conform to the skin and reduce noise during the signal acquisition without inducing pain during its removal. Furthermore, PUD/PEDOT:PSS electrodes showed stretchability and a Young’s Modulus similar to skin, which demonstrates the durability of the electrode and tolerance to repeated mechanical strains, while maintaining high conductivity. Strain-stress, cyclic stress, electrotensile as well as adhesion tests confirm this PUD/ PEDOT:PSS composite’s excellent mechanical properties. Weeklong EMG and ECG measurements with the same electrodes also showed its capability for long-term usage.
Such an electrode is more suitable for long-term health monitoring of patients with fragile skin, such as premature infants.

The conducting polymer poly(3,4-ethylenedioxythiophene) (PEDOT) is a promising material for improving the stimulation efficiency of neural microelectrodes due to its many advantageous characteristics, such as mechanical compliancy, electrochemical stability, and high conductivity. Studies on the long-term in vivo electrochemical stability of penetrating PEDOT-coated electrodes undergoing high-frequency stimulation are not extensively done and the immune response of the brain towards PEDOT-coated stimulating neural probes is not well investigated. In this study, electropolymerized PEDOT doped with tetrafluoroborate (PEDOT:BF4) is selectively deposited on the electrodes of platinum iridium (PtIr) neural probes and implanted for 2 weeks and 2 months to evaluate the effect of implantation on the electrical performance, along with the foreign body response to the probes. Histological analysis after 8 weeks of implantation shows no difference in the degree of inflammation around PtIr and PEDOT probes. Furthermore, PEDOT and PtIr probes are implanted for 60 days, and subject to daily high frequency stimulation while being monitored for changes in electrochemical properties. Impedance measurements confirm an overall lower impedance for PEDOT probes. This study shows that PEDOT:BF4 coatings can contribute to the electrochemical stability of neural interfacing devices for stimulation and recording.

Current approaches in the healthcare field use electrodes as biomedical devices at the interface of biological systems and electronics to either transmit electrical impulses or as sensors to record bioelectric signals. The main hindrance to these measures is the resistance to the flow of the electric signal from the living tissues, called the impedance. Recent advances are proposing organic conducting polymers, mainly poly(3,4-ethylenedioxythiophene) (PEDOT), to improve signal acquisition and lower the impedance at the electrode/tissue interface. In fact, PEDOT has been shown to have a low impedance, a high conductivity due to its mixed electronic ionic conduction, good electrochemical stability, and biocompatibility allowing its use in bioelectronic devices.¹ The present study proposes using PEDOT as a coating on Nickel plated copper (Ni-Cu) electrodes to design innovative devices with low impedance and high conductivity to record bio-signals for diagnosis, monitoring, and prevention of medical conditions. The nickel coating has been achieved through an electroless plating process in a nickel-plating bath.² Afterwards, the PEDOT coating has been achieved through cyclic voltammetry (CV) and a galvanostatic chronopotentiometry (CP) electropolymerization of the 3,4-ethylenedioxythiophene (EDOT) monomer. The bath contained the EDOT monomer and the dopant, lithium perchlorate (LiClO₄), dissolved in either water or acetonitrile (ACN). A variety of potential ranges were considered for cyclic voltammetry, but only cycling held constant between -1.0 V and +1.2 V produces the agglomeration of PEDOT on the substrate. The chronoamperometric method resulted in homogeneous/uniform PEDOT films, and by optimizing the electropolymerization conditions, we demonstrated the mechanical and electrochemical stability of PEDOT doped with LiClO₄ deposited on Ni-Cu electrodes. The coatings have been analyzed and characterized using electrochemical impedance spectroscopy (EIS) and CV in a phosphate-buffered saline solution (PBS), X-ray photoelectron spectroscopy (XPS), and scanning electron microscopy (SEM). As a result of PEDOT coating’s potential electrocatalytic properties, an improvement in the electrode’s electrochemical properties was achieved, resulting in reduced impedance and higher performance. Due to their high capacitance and low impedance, PEDOT Ni-Cu electrodes can be used as convenient electrodes for detecting biological signals, improving biomedical devices in the future.

References:
1. Rossetti, N., Luthra, P., Hagler, J. E., Jae Lee, A. H., Bodart, C., Li, X., ... & Cicoira, F. Poly (3,4-ethylenedioxythiophene)(PEDOT) coatings for high-quality electromyography recording. ACS Applied Bio Materials. 2019, 2(11), 5154-5163.

2. Son, J., Hong, Y., Yavuz, C. T., & Han, J. I. Thiourea-based extraction and deposition of gold for electroless nickel immersion gold process. Industrial & Engineering Chemistry Research. 2020, 59(16), 8086-8092.

Solution-processable organic materials are desirable for functionalized wearable devices. To maximize that feature of organic polymers, printing is one of the best methods to cast films or patterns. Herein, we fabricated fully-stretchable organic electrochemical transistors(OECTs) by a conventional PBC printer. To achieve the stretching of the whole body of the devices, a printed planar gate electrode and polyvinyl alcohol(PVA) hydrogel electrolyte were employed. The simplicity of the PCB printer made it possible to print a wide range of viscous inks, from an organic semiconductor to a hydrogel precursor. A Stretchable silver paste provided a soft feature to drain/source, gate and interconnect without any strategies to improve the stretchability of metallic components, such as pre-stretching methods and formation of stretchable patterns. Moreover, unlike reported studies of printed ion-gel electrolytes for transistors, a printed hydrogel electrolyte on OECTs has not been reported yet. The resulting OECTs showed decent performance comparable to ink-jet or screen-printed OECTs; the maximum transconductance and on/off ratio were 1.2mS and around 1400, respectively. The devices were stable in electromechanical behavior until 30% strain which is required for wearable electronics on the skin.

Microelectronics has transformed our lives. It has changed the way we collect, process, and transmit information. The intersection between microelectronics and biology has also been transformative – ionic currents that control cardiovascular and neural systems are detected and even corrected using electronics (e.g., EKG & defibrillators). Yet, the microelectronics world has barely “sampled” the vast repertoire of chemical information in our biological world. Take for example the human immune, endocrine, and gastrointestinal systems – they are largely opaque to the methods of electrical sensing and communication. In biology, information is often contained in the structure of its molecules – molecules that move from place to place and based on their structure, convey information and provoke a response.

We envision new processes and deployable products that open the dialogue between biology and microelectronics – that eavesdrop on and manipulate biological systems within their own settings and in ways that speed corrective actions. We view biofabrication and synthetic biology as integral technologies for achieving this vision. Synthetic biology, often visualized as an innovative means for “green” product synthesis through the genetic rearrangement of cells, can also provide a means to connect biological systems with microelectronic devices. Cells can be reprogrammed to close the communication gap that exists between the electrons and photons of devices and the molecules and ions of biology. This is enabled, in part, through redox mediators – biological carriers of electrons that transfer “packets” information to and from electronics. We have suggested the purposeful electronically actuated elicitation of gene expression is “electrogenetics”. Biofabrication, the assembly of biological components using biological means or mimics thereof, offers a means to close the fabrication gap – a gap that stems from the disparity between biological systems, assembled of labile components using built-in error correction, and devices, built of potentially toxic materials using error prevention and byproduct exclusion. Here, innovative materials, electronics, biomolecular and cellular engineering strategies can be developed to mediate “molecular” communication - information transfer to microelectronic systems and back. New systems and devices are continually emerging that integrate abiotic and biological components (e.g, animal-on-a-chip devices, chip based manufacturing systems, etc.) at a hierarchy of length scales. New systems may emerge that eavesdrop on and electronically guide cellular consortia, vastly expanding our synthetic biology repertoire while utilizing increasingly complex raw materials.

We suggest that a great many of our society’s grand challenges in sustainability, food, energy, and medicine may be addressed by developing tools that open lines of communication between the biological and electronics worlds.

A novel approach for bacteria biosensing using organic electrochemical transistors (OECT)¹ has been demonstrated in the sensing of cells and metabolic products (carbohydrates, protein, and cations)². OECTs are attractive for bacteria biosensing due high sensitivity, fast diagnostics, and simpler bacteria handling. The use of OECT enables the detection of bacterial contamination in home-liquid-goods and provide potential real-time monitoring capabilities. Two main requirements for bacteria sensing in liquid products are: (a) non-specific detection (no capture agents) - there are hundreds of common bacteria strains present in daily living that are harmful to humans; (b) high sensitivity – for early detection of liquid goods contamination, at bacteria concentrations as low as a few hundred colony-forming units per mL (CFU/ml). OECTs can respond to bacteria attached to (or near) either the source-drain channel or the gate electrode. Here, we report on universal bacteria sensing using OECTs integrated within a microfluidic flow cell. A conductive microporous filter membrane in the flow cell device was utilized to accumulate the bacteria and serve as a gate electrode for OECT operation (“gate filter”). The device can detect ~ 10² CFU/ml bacteria in mixtures of household liquid products and common bacteria growth broths. This is the result of bacteria accumulation on the gate causing a shift in the OECT source-drain channel current (I_ds).

The overall sensor consists of a dual PEDOT: PSS gate OECT, a 3D-printed flow cell, and an Au-coated 0.4um PETE filter membrane as the gate electrode. The dual OECTs are integrated into the flow cell device and both channel sensing and gate sensing approaches are evaluated using pseudomonas fluorescens, a common Gram-negative bacterium. OECT channel sensing shows good discrimination between bacteria and sterile solutions, with higher bacteria concentration resulting in higher I_ds shift and increased peak transconductance (g_m). At V_g = 0.3V, compared to sterile solution a solution of 10⁵ CFU/ml bacteria results in ~ 43% and 100% increase in I_ds (2.51mA) and g_m (7.2mS), respectively. In the flow cell device operation, bacteria trapped on the gate filter results in additional gate-to-electrolyte potential drop on the effective gate voltage. Sterile and bacteria solutions of 10² and 10³ CFU/ml display the same I_ds starting point (4.6 mA) at V_g = 0V. As gate bias is applied, the higher bacteria concentration samples have a sharper I_ds reduction. At V_g = 0.3V, the channel current is reduced to 1.7 mA for the sterile solution, 1.3 mA for the 10² CFU/ml, and 0.5 mA for the 10³ CFU/ml bacteria solutions. The higher bacteria concentration results in a smaller voltage required to switch off the OECT of 0.5 and 0.7V for 10³ and 10² CFU/ml, respectively. With the OECT flow cell approach, bacteria detection resolution reached a low value of ~ 10² CFU/ml. For continuous real-time detection testing using OECT flow cells, the device maintains I_ds = 3.5mA at V_g = 0.3V when flowing sterile solution at 3ml/hr. Introducing 10³ CFU/ml bacteria solution results in the I_ds current decrease with a 1.5mA/hr rate. In summary, the 3D-printed flow cell incorporating OECTs has been used to sense low levels of bacteria present in commercial household liquid products within an hour without the need for amplification, providing a promising approach towards future biosensing system.

Frantz, E., Han, D., & Steckl, A. ECS Meeting Abstracts, 2021, May, No. 55, p. 1416.
Marks, A., Griggs, S., Gasparini, N., & Moser, M. Advanced Materials Interfaces, 2022, 9(6), 2102039

The existence of an electronic coupling between electrogenic cells and electronic supports is the bases of any tailored system employed to study biology-driven phenomena. During real-time monitoring of electrophysiological signals (action potentials, APs), the communication typically occurs at the cell (neurons or cardiomyocytes)/transducer interface, whose optimization is fundamental to make the investigation of neurodegenerative and cardiac diseases effective. In the last 20 years, organic materials have been largely used for the fabrication of bioelectronic devices thanks to their biocompatibility, easy processability, tunability of physical properties, stimuli-responsiveness and high conformability ¹.
Organic electrochemical transistors (OECTs) using Poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) PEDOT:PSS as active material, present features that make them perfectly suitable for the record of electrophysiological potentials: i) the biocompatibility, ii) the flexibility, and iii) the double ionic/electronic conductivity of the polymer. The cell/transducer interface is, in this case, represented by the active material that interfaces the biological system and dictates the transduction of the ionic transmembrane flows (able to dope/de-dope the polymer) into an electrical readout ². Moreover, iv) the high source-drain current levels and v) the large transconductance values that characterized this type of devices, allow to keep low operating voltages and reduced heat generation, necessary for the system durability in aqueous biological environment ³. Being a non-invasive measurement, it increases the vitality of the cells, allowing multiple and long-duration recordings, fundamental for chronic studies.
In this work, we report in vitro recordings of APs of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) by using planar OECTs with spin-coated PEDOT:PSS channel. hiPSCs-CMs were cultured directly on the polymeric surface. Extracellular potential recordings were obtained by simply turning on the transistors at their maximum transconductance and following the variation of both the drain-source and the gate-source currents. Extracellular signals present a typical and predominant negative phase (with amplitude around 0.3 mV) and an expected short duration of around 20 ms ^2,4. Intracellular signals were recording adopting a cell optoporation protocol (large pulse, low current intensity) that enabled the measurement without stimulating the cells ⁵. Amplitudes in the range of 3-4 mV were recorded, which is comparable to performance obtained with similar devices that use electroporation ³. To improve the photocurrent response different formulations of PEDOT:PSS with the addition of gold nanoparticles are tested. Drug screening tests are performed to assess the ability of the devices to detect cardiotoxic effects; live/dead assay and immunofluorescence imaging to evaluate their biocompatibility. In future, additional electronics and biosensing elements could be complemented on the same device paving the way to multifunctional, reliable, and low-cost biosensors.

1. Ohayon, D. & Inal, S. Organic Bioelectronics: From Functional Materials to Next-Generation Devices and Power Sources. Adv. Mater. 32, 2001439 (2020).
2. Gu, X. et al. Organic Electrochemical Transistor Arrays for In Vitro Electrophysiology Monitoring of 2D and 3D Cardiac Tissues. Adv. Biosyst. 3, 1–8 (2019).
3. Jimbo, Y. et al. An organic transistor matrix for multipoint intracellular action potential recording. Proc. Natl. Acad. Sci. 118, 1–8 (2021).
4. Liang, Y. et al. Tuning Channel Architecture of Interdigitated Organic Electrochemical Transistors for Recording the Action Potentials of Electrogenic Cells. Adv. Funct. Mater. 29, (2019).
5. Abbott, J. et al. A nanoelectrode array for obtaining intracellular recordings from thousands of connected neurons. Nat. Biomed. Eng. 4, 232–241 (2020).

Electrochemical detection methods are promising for accessible biosensors to all citizens because of their simple principle, and various types of electrochemical biosensors have been developed, including wearable sensors and implantable sensors [1]. However, these sensors cannot reach wide-area spatial information, although they can detect biological information near the electrodes. Therefore, we are proposing a new-type biosensor, an autonomous mobile biosensor that moves in solution and continuously tells us the biological information.
In the Marangoni motor system, the motor releases surfactant from its body, allowing it to move in solution while generating a surface tension gradient around itself [2]. Urease, one enzyme, catalyzes the production of ammonia molecules from the biomolecule urea, and the ammonia reduces surface tension [3]. It suggests that the urease-modified motor can generate driving force in urea containing bio-solution without external energy supply. In addition, conductive polymer hydrogels are suitable for the motor body material because of their conductivity, which mediates electrochemical information, and their porosity, which allows enzymes to be carried in the three-dimensional polymer network. Common electrochemical sensors use wires to connect the sensing electrodes with external measurement systems for the collection of electrical signals, but the wires inhibit the movement of the motors in the autonomous mobile biosensor. In this study, a wireless sensing system was developed to acquire biological signals from the self-propelled conductive hydrogel motor.
The conductive hydrogels were fabricated from a mixture of poly(3,4-ethylene dioxythiophene): poly (styrene sulfonate) (PEDOT: PSS) and dimethyl sulfoxide (DMSO) [4]. Enzyme-based motorization was investigated via modification of urease on a conductive hydrogel by physical cross-linking with glutaraldehyde. In developing wireless motor detection, we examined impedance sensing methods. Two parallel Au electrodes, called direct electrodes (DEs) were placed in PBS and connected to the electrochemical analyzer. In addition, four materials (Au, ITO; indium tin oxide, Gel; conductive hydrogel, glass), called bipolar electrodes (BPE) were placed between the DEs wirelessly, and the impedance changes depending on the material and placement of the BPEs were investigated.
As a result, the urease-modified hydrogel motor began moving in PBS including urea on the basics of the enzymatic reaction. It indicates that urease-modified gel can obtain driving energy from urea and move continuously in bio solution. In addition, the impedance change was studied depending on the wireless BPE material, and it was found that when conductive materials (Au, ITO, Gel) were put between the DEs, the impedance at high frequency become lower than when no material was put in the solution. In contrast, when glass, an insulator, is placed between the DEs, the impedance increases. It indicates that the conductivity of the BPE material alters the current flowing between the DEs. Besides, impedance change dependent on the wireless gel position was also investigated, and it was found that the impedance change became smaller as the gel moved away from the DEs. It indicates that the impedance measurement method provides information on the movement of the gel wirelessly. In the future, we will capture urea concentration-dependent changes in gel motion wirelessly and investigate potential applications in biosensing.

[References]
[1] S. Himori and T. Sakata, RSC Adv., 12, 5369–5373, (2022). [2] X. Arqué et al., Nat. Commun., 10, 2826, (2019). [3] Y. Xu et al., Chem. – An Asian J., 16, 1762–1766, (2021). [4] B. Lu et al., Nat. Commun., 10, 1043, (2019).

Microbial extracellular electron transfer (EET) by electroactive bacteria can be reprogrammed using bioengineering to enhance existing technologies (e.g. microbial fuel cells) or create entirely new ones (living electronic sensors). A current bottleneck in engineering electroactive bacteria is the selection and development of chassis strains that can both host complex EET machinery and survive under conditions relevant to the application of interest. In this talk I will describe our efforts to develop Marinobacter atlanticus, a gram-negative marine bacterium, as an engineered electroactive bacterium for sense and respond in seawater. Genetically encoded sensors optimized for use in E. coli were used to control expression of the Shewanella oneidensis electron transfer pathway in planktonic and biofilm attached cells. Electrical current production could be induced during biofilm growth in artificial seawater when sensors controlling expression of different components of the pathway were activated by addition of 2,4-diacetylphloroglucinol (DAPG), isopropyl β -d-1-thiogalactopyranoside (IPTG), and tetracycline (Tc). Significant current production also required addition of menaquinone, which M. atlanticus does not produce, for electron transfer from the inner membrane to the expressed electron transfer pathway. Electron transfer was also reversible, indicating electron transfer into M. atlanticus could be controlled. These results show that an operationally relevant marine bacterium can be genetically engineered for environmental sensing and response using an electrical signal.

Photosynthetic microorganisms and their molecular components represent attractive functional elements for harvesting and conversion of solar energy in bioelectronic devices [1].
We have recently investigated several approaches for addressing the Reaction Center (RC) photoenzyme extracted from the purple non sulfur photosynthetic bacterium Rhodobacter (R.) sphaeroides in optoelectronic devices for conversion of solar energy into photocurrent.
We have used polydopamine (PDA) for immobilizing and interfacing RC on ITO electrodes, by simple polymerization of dopamine in aqueous media [2].
We also used polydopamine to directly integrate living cells of photosynthetic bacteria onto electrodes in bio-photoelectrochemical cells. Two different approaches were used for R. sphaeroides and its closely related strain R. capsulatus, respectively. The encapsulation of
entire bacterial cells of R. sphaeroides in PDA conductive coating was obtained by addition of dopamine in bacterial growth medium [3]. In the case of the R. capsulatus a one-pot procedure was used, which was initiated by oxygenic polymerization followed by electrochemical polymerization of dopamine, including redox mediators [4].
We have also explored the possibility to directly interface photosynthetic microorganisms with electrodes without the use of an additional soft interfacing material. For this purpose, we used the unicellular diatom algae Phaeodactylum tricornutum, exploiting their spontaneous biofilm formation.
Our study discloses new concepts for the generation of bio-hybrid materials for sunlight photoconversion and for light-responsive bioelectronics from the combination of photosynthetic microorganisms with tailored functional materials.

[1] F. Milano, A. Punzi, R. Ragni, M. Trotta, G.M. Farinola, Adv. Funct. Mater., 29, 1805521, (2019).
[2] M. Lo Presti, M. M. Giangregorio, R. Ragni, L. Giotta, M. R. Guascito, R. Comparelli, E. Fanizza, R. R. Tangorra, A. Agostiano, M. Losurdo, G. M. Farinola, F.Milano, M. Trotta, Adv. Electron. Mater., 6, 2000140, (2020).
[3] D. Vona, G. Buscemi, R. Ragni, M. Cantore, S. Cicco, G.M. Farinola, M. Trotta, MRS Advances, 5(18-19): p. 957-963, (2020).
[4] G. Buscemi, D. Vona, P. Stufano, R. Labarile, P. Cosma, A. Agostiano, M. Trotta, G.M. Farinola, M. Grattieri, ACS Appl. Mater. Interfaces 14, 23, 26631–26641, (2022).

A fundamental understanding of the extracellular microenvironments of O₂ and reactive oxygen species (ROS) such as H₂O₂, ubiquitous in microbiology, demands high-throughput methods of mimicking, controlling, and perturbing gradients of O₂ and H₂O₂ at microscopic scale with high spatiotemporal precision. However, there is a paucity for a high-throughput strategy of microenvironment design and it remains challenging to achieve O₂ and H₂O₂ heterogeneities with the microbiologically desirable spatiotemporal resolutions. Here we report the inverse design, based on machine learning (ML), of electrochemically generated microscopic O₂ and H₂O₂ profiles relevant for microbiology. Microwire arrays with suitably designed electrochemical catalysts enable the independent control of O₂ and H₂O₂ profiles with spatial resolution of ~10¹ μm and temporal resolution of ~10⁰ sec. Neural networks aided by data augmentation inversely design the experimental conditions needed for targeted O₂ and H₂O₂ microenvironments while being order-of-magnitude faster. Interfacing ML-based inverse design with electrochemically controlled concentration heterogeneity creates a viable fast-response platform towards better understanding the extracellular space with desirable spatiotemporal control.

Due to the anthropogenic CO₂ emissions, the atmospheric concentration of CO₂ exceeded 413 ppm in 2020, sharply increasing from 270 ppm at the beginning of the industrial revolution. Dramatic climate changes, such as extreme heat waves, wildfires, and droughts, are affecting human societies and ecosystems at such alarming levels that their influence can no longer be ignored. Thus, developing alternatives to fossil fuels and mitigating CO₂ emissions at a necessary scale is more urgent than ever to establish a carbon-neutral society. Catalytic CO₂ conversion to renewable fuels has been the focus of intensive research with the goal of closing the carbon loop. Photosynthetic biohybrid systems (PBSs), in which a photoactive semiconductor directly interfaces with nonphotosynthetic, CO₂-reducing bacteria, represent a promising approach for the renewable and sustainable production of fuels and chemicals. They combine the best features of semiconducting nanomaterials and biological whole-cell catalysts to power biocatalytic CO₂ conversion with light. Semiconducting nanomaterials can harvest solar energy efficiently. Nature’s catalytic machinery enclosed in its cellular environment provides low activation energies and exquisite product selectivity through its cascade reactions. We demonstrate that by tunning the local chemical environment of a silicon nanowire – acetogen sporomusa ovata (S. ovata) biohybrid, we can maximize the interfacial area between living cells and cathodes and boost the bioelectrochemical CO₂ reduction rate. In addition to optimizing the local environment, the rational design of biocatalysts in PBSs is imperative to reduce CO₂ into valuable chemicals effectively. We introduce a methanol adapted S. ovata to enhance the sluggish biocatalytic activity of wild-type microorganisms into our silicon nanowires (Si NWs) photocathodes for efficient solar-to-chemical conversion. Electrochemical impedance spectroscopy (EIS) is exploited to study the biohybrid system and the biofilm cathodes. We illustrate the fundamental charge transfer mechanisms at the biotic-abiotic interface between silicon nanowires and microorganisms. Using adapted S. ovata decreases the charge transfer resistance at the cathodes and facilitates charge transfer from directly interfaced solid electrodes. Finally, this new generation of biohybrids with adapted S. ovata enables an increase of solar CO₂ fixation under photoelectrochemical reducing environments on the Si NWs photocathode.

An enzymatic biofuel cell (EBFC) is a type of power generating device where enzymes function as catalysts to oxidize biofuel (saccharides such as glucose or alcohols)^[1]-[4] and reduce oxygen molecules.^[5]-[7] It can be operated under mild conditions of neutral solution, room temperature and atmospheric pressure. The main advantage of an EBFC is the utilization of renewable energy sources through enzymatic reactions. Among the fuel sources currently available for EBFCs, formic acid is oxidized by formate dehydrogenase (FDH) at a low potential and is expected to high-voltage EBFCs. Most of the previously reported fuel cells with formic acid as the fuel utilize palladium or platinum as catalysts, and there are few reports of enzymes being used as catalysts. Using enzymes has the advantage of being relatively inexpensive compared to other catalysts in EBFCs. In this study, we focused on FDH with nicotinamide adenine dinucleotide (NAD) as the active site and prepared enzymatic bioanodes modified with three different electropolymerized thiazine dyes, methylene blue (MB), thionine (Th), and Azure A (AA), as electrocatalyst and investigated their electrochemical properties.

The bioanodes were prepared as follows: 1) carbon powders (carbon nanotube or sucrose-derived carbon) was dispersed in water with sodium polyglutamate as the binder and dropped on carbon-felt (CF) electrodes (thickness: 2 mm; diameter: 11 mm), then the carbon electrodes were modified with the three electropolymerized films, polyMB, polyTh, and polyAA, and 3) FDH solution was drop-cast on the electrodes. Electrochemical measurements were conducted at room temperature in the phosphate buffer solution (0.1 mol dm^-3 PBS at pH = 7.0,) containing NAD (10 mmol dm^-3) and sodium formate (0.15 mmol dm^-3).
At the prepared electrode, formate was anodically oxidized with FDH at the electropolymerized electrode, and the coenzyme NAD was simultaneously reduced to NADH. The oxidation of NADH by the electropolymerized thiazine was expected to proceed rapidly. We successfully observed an oxidation current at −0.05 V (vs. Ag/AgCl), generated by the anodic decomposition of formate ions, in the substrate solution. This confirms that the electrochemically deposited thiazine polymer can act as electrocatalyst for the series of above oxidation reaction as expected. Among three thiazine dyes, it was found that polyMB has the highest catalytic activity for NADH oxidation, yielding the highest oxidation current densities owing to the efficient electron transfer process. The comparison of the electrochemical properties of the bioanode prepared with CNT and pyrolytic sucrose-derived carbon will also be discussed in the presentation.

References
[1] S. Komaba, et al., Electrochemistry, 76, 55 (2008)
[2] Y. Handa, S. Komaba, et al., ChemPhysChem, 15, 2145 (2014).
[3] R. Yasujima, S. Komaba, et al., ChemElectroChem, 5, 2271, (2018).
[4] R. Toda, S. Komaba et al., ChemElectroChem, 8, 4199 (2021).
[5] K. Yamagiwa, S. Komaba, et al., J. Electrochem. Soc., 162, F1425 (2015).
[6] K. Yasueda, S. Komaba, et al., J. Electrochem. Soc., 165, F1369 (2018).
[7] R. Tatara, S. Komaba, et al., J. Electrochem. Soc., 168, 074506 (2021)

Cable bacteria, a group of filamentous electroactive bacteria, have developed a unique energy metabolism and parallel fibre structures demonstrating electron transport for conduction lengths up to 1 cm and with fibre conductivities exceeding 10 S/cm. Conduction measurements carried out in high vacuum excluded the possibility of ionic conduction, but the fundamental charge transport mechanisms remain unknown. The observed electron transport in cable bacteria over distances in the order of centimeters is remarkable in the biological world.

Cable bacteria as ‘living electrical wires’ are of fundamental interest to better understand the underlying biological processes, but are also potentially interesting as alternative organic electronic materials for the emerging field of bioelectronics as e.g. biocompatible electrical connections and circuits, conductive composite materials, (nano-) sensors, transistors,... In order to investigate the intrinsic electrical properties and underlying transport mechanisms, our approach is to study them with a range of macroscopic and nanoscale solid state electrical characterisation techniques, in combination with structural and analytical techniques ranging from SEM to ToF-SIMS.

The electrical circuitry in a single cable bacterium is visualized with nanoscopic resolution using conductive atomic force microscopy (C-AFM). Combined with perturbation experiments, it is demonstrated that electrical currents are conveyed through a parallel network of conductive fibers embedded in the cell envelope, which are electrically interconnected between adjacent cells. This structural organization provides a fail-safe electrical network for long-distance electron transport in these filamentous microorganisms.

Impedance spectroscopy provides an equivalent electrical circuit model, which demonstrates that dry cable bacteria filaments function as resistive biological wires. Temperature-dependent electrical characterization reveals that the conductivity can be described with an Arrhenius-type relation over a broad temperature range (-195°C to +50°C), demonstrating that charge transport is thermally activated with a low activation energy of 40-50 meV. Furthermore, cable bacterium filaments can be utilized as the channel in a field-effect transistor.

The measured intrinsic electrical properties result very similar to for instance organic semiconductors, situating them in the context of electrical functional materials between semiconductors and conductors. These electrifying ‘bad guy’ microorganisms – with from various perspectives behave as they are not supposed to – are attracting growing interest and in the long-term could open novel avenues in emerging domains such as bioelectronics, biodegradable electronics and electronic biological materials (e-biologics). As a proof-of-principle for electrical transmission capabilities through cable bacteria filaments, the music track “Bad Guy” of Billie Eilish is transported through them. Cable Bacteria are rock & roll.

Microbial electrosynthesis – using renewable electricity to stimulate microbial metabolism – holds the promise of sustainable chemical production. A key limitation hindering performance are slow electron transfer rates at biotic-abiotic interfaces. Here, we rationally design a water-processible n-type organic semiconducting polymer and demonstrate its use as a soft conductive material to encapsulate electroactive Shewanella oneidensis MR-1. The self-assembled three-dimensional living biocomposite amplifies current uptake from the electrode ≈674-fold over controls with the same initial number of cells, thereby enabling continuous synthesis of succinate from fumarate. By demonstrating synergy between living cells with n-type organic semiconductor materials, these results provide new strategies for improving the performance of bioelectrosynthesis technologies.

In bioelectrochemical systems (BESs), anaerobic conditions are used to generate current from microorganisms because oxygen can compete with an electrode as a terminal electron acceptor. However, prior studies have shown that oxygen enhances current production from Shewanella oneidensis MR-1 by counterbalancing a modest decrease in current per cell with a substantial increase in the total biomass. This opens the possibility that BESs based on oxygen-tolerant microorganisms might not need anaerobic conditions for maximal current production. Here we investigated this question in the facultative anaerobe Escherichia coli, which has been engineered to produce current using the Mtr pathway from Shewanella oneidensis MR-1.
To investigate the effect of oxygen on the current production of Escherichia coli, we have introduced different oxygen levels to BESs containing Mtr-expressing E. coli and monitored current production, biomass, protein expression and metabolic products. Our results showed that oxygen exposure substantially boosted current up to 30-fold relative to anaerobic conditions. With increasing oxygen levels, both the current per cell and total biomass increase. Oxygen enhances the expression of the Mtr proteins relative to anaerobic conditions. Additionally, oxygen levels linearly increased the abundance of the first protein in the Mtr pathway, indicating that this protein is the limiting step in the current production. Additionally, oxygen increased energy flux and substrate utilization.
In summary, oxygen enhances the current production of Mtr-expressing Escherichia coli. This work challenges the prevailing paradigm that anaerobic conditions promote current production, suggesting that bioelectronic applications can be performed in oxygen-rich conditions.

Electrolyte-gated transistors (EGTs) based on organic semiconductors and graphene derivates are emerging in the field of biosensing because they are ultrasensitive, label-free, can be fabricated on flexible substrates at low cost and interfaced with biological samples.^1,2 We show some examples to demonstrate the possibility to use this technology to develop biosensors for different healthcare applications, by just selecting the most effective device architecture and material.
All the presented devices share a common transistor architecture, consisting of two interdigitated electrodes, source and drain, covered with an active material, in contact with a gate electrode through an electrolyte. The biosensing event takes place at the gate/electrolyte interface, by functionalization of the gold gate with a biorecognition moiety, and it is amplified by the active material channel thanks to the high-capacitance electrical double-layers formed at the gate/electrolyte and electrolyte/channel interfaces.
We fabricated EGTs biosensors for ultrasensitive detection of biomarkers by using different materials in the channel, namely ambipolar Reduced-Graphene Oxide, organic p-type semiconductor TIPS-Pentacene and poly(3,4-ethylenedioxythiophene)polystyrene sulfonate (PEDOT:PSS) polymer mixture.
Thanks to tailored gate bio-functionalization, we realized a disposable rGO-EGT immunosensor for the detection of anti-Infliximab antibodies, which are produced by patients upon treatment with the immunotherapeutic drug Infliximab and can make the therapy ineffective. Furthermore, we developed an Electrolyte-Gated Organic Field-Effect Transistor (EGOFET) immunosensor for the detection of anti-Nivolumab antibodies, based on the organic semiconductor TIPS-pentacene, showing fM theoretical Limit of Detection (LOD).³ We also successfully employed an organic electrochemical transistor (OECT) genosensor for the detection of oligonucleotides, thanks to a facile functionalization process based on polydopamine.⁴ Finally, we demonstrated the possibility to use EGOFET for the specific detection of transcription factors, by using the consensus DNA sequence as biorecognition element.
We conclude that EGTs biosensors show promising performances for the future application at the Point-of-Care but also for fundamental studies on macromolecules interactions.


References
1) Burtscher, B.; Manco Urbina, P. A.; Diacci, C.; Advanced Healthcare Materials. 2021,10 (20), 2100955.
2) Torricelli, F.; Nat. Rev. Methods Prim. 2021, 1 (66).
3) Sensi, M.; Chem. Commun. 2021, 57 (3), 367–370.
4) Sensi, M.; Macromol. Mater. Eng. 2022, 307 (5), 2100880.

Bacterial infections that do not respond to treatment are a leading cause of death worldwide and a major public health threat. For example, the O157:H7 strain of Escherichia coli (E. coli) is considered one of the most dangerous foodborne pathogens. It is crucial to detect pathogenic organisms at an early stage so that appropriate treatment and targeted use of antibiotics can be undertaken. Conventional detection techniques, such as enzyme-linked immunosorbent assay (ELISA) or polymerase chain reaction (PCR) are time-consuming, expensive, and require trained professionals. A method for rapid detection of bacteria that is inexpensive, provides a reliable result, and is easy to use is becoming increasingly important. The development of suitable electronic transducers that provide label-free, low-cost, rapid detection of bacteria is of great interest, as this approach could be extended to the development of handheld devices for point-of-care applications.

In previous work, a detection method based on an extended-gate field-effect transistor (EGFET) was developed for the detection of E. coli K12, a common well investigated apathogenic model strain. The sensing region of the EGFET was functionalized with modified porphyrins containing two different linkers: One linker connects the porphyrin to the electrode surface of the gate, and the other binds bacterial cells to the functional layer via a specific peptide chain. After applying a bacterial suspension onto the functional layer, the cells can thus bind to it. The negative charge on the surface of the cells changes the electrical potential at the electrode surface, which affects the electrical behavior of the EGFET. The presence of bound E. coli cells on the functionalized sensor surface has already been successfully detected¹.

The aim of this research is to concentrate bacteria specifically on the sensor surface of the EGFET by exerting a dielectrophoretic force on the cells in a combined system. The electrokinetic effect of dielectrophoresis (DEP) enables contactless manipulation of biological cells in fluids. DEP utilizes the geometry of electrodes to generate non-uniform high-frequency electric fields that exert an attractive or repulsive force on dielectric particles, inducing movement toward areas of maximum electric field gradient (positive-DEP) or minimum electric field gradient (negative-DEP). DEP is a flexible and versatile method for immobilizing, separating, and concentrating cells and has been successfully optimized for the fixation of bacteria on electrodes in previous work. It will be investigated to what extent the reliability and sensitivity of the sensor can be increased by dielectrophoretically trapping the bacteria on the transistor gate. Furthermore, it is possible to extend the functionality of the sensor with selectivity by combining it with DEP. DEP is capable of selectively separating biological particles by properties such as size, material, and shape, which allows selective detection of a bacterial species from a heterogeneous suspension.

Reference: 1. Könemund, L., Neumann, L., Hirschberg, F. et al. Functionalization of an extended-gate field-effect transistor (EGFET) for bacteria detection. Sci Rep 12, 4397 (2022)

Key words: dielectrophoresis (DEP), biosensing, EGFET, porphyrin, E. coli immobilization

Recently printing techniques attracted significant attention for the fabrication of electronic devices especially organic electrochemical transistors (OECTs). Printing method is a low-cost additive process with fast prototyping and compatibility with different flexible or stretchable substrates. Poly(3,4-ethylenedioxythiophene) : polystyrene sulfonate (PEDOT:PSS) is a commonly used p-type material for OECTs due to its stability in aqueous media. Most of the reported OECTs to date have large channel lengths in tens of micrometers, which limits the transistor performance and transconductance. Downscaling the channel length is challenging by a common photolithography method. Here we used the printing technique to fabricate vertical configuration of OECTs where the channel length is defined by the thickness of printed active material (PEDOT:PSS), sandwiched between source and drain electrodes (silver electrodes). Printable semi-solid gel is used as an electrolyte for devices. We compared the planar configuration with vertical devices, and significantly higher transconductance is obtained for vertical configuration.

Bioelectrical signalling drives behaviour of cells at scales from single bacteria to multicellular tissues. Improved understanding of bioelectrical phenomena, including their influence on immune cell function, will lead to improved electroconductive materials relevant to repair of epithelial and tissue damage.
When an epithelium is wounded an endogenous, steady electric field (EF) of ~100 mV/mm is driven through the neighbouring tissues (cathode at the centre) and the success of wound closure relates to the presence and magnitude of this wound EF. In part this is because the EF steers migration of epithelial cells toward the wound site to close the gap. But Immune cells, including monocytes and macrophages, are also important contributors to successful wound healing. For many years the stimuli driving their function were principally attributed to chemical signalling factors present within the wound microenvironment, with no regard given to bioelectrical signals.
We therefore studied the responses of cultures of primary human monocytes and macrophages exposed to physiologically relevant EFs. Time lapse observation of monocytes, which are macrophage precursors, showed that an EF of 150 mV/mm applied for 2h steered migration toward the cathode and increased the speed of migration compared to controls (no EF). This suggests that these ‘first responders’ would be guided toward the wound centre (cathode) by an endogenous EF.
However, human macrophages responded differently. Macrophages migrated preferentially toward the anode (opposite to monocytes) and their speed of migration was increased compared to controls. Impressively, the macrophages were exquisitely sensitive to the EF stimulus, responding with migration toward the anode at EFs as small as 5 mV/mm, well below the threshold of endogenous wound EFs. This suggests that macrophages arriving, later than the monocytes, migrate to the wound edges (anode) to clear debris and microbes.
Directed migration of macrophages was associated with elongation of the cells parallel to the EF axis and reorganisation of the actin cytoskeleton toward the leading edge. Since a key role of macrophages is phagocytic uptake of microbes and apoptotic cells in the wound environment, we tested whether the EF also enhanced phagocytosis. Macrophages were exposed to an EF for 2h then carboxylate beads, Candida albicans, or apoptotic neutrophils were added to the cultures and left without any EF for a further 2h. Phagocytosis increased significantly in each case, demonstrating that the EF ‘priming’ improved phagocytotic efficiency.
Mechanistically, these EF-induced functional changes are accompanied by clustering of phagocytic receptors toward the anode, enhanced PI3K and ERK activation, and mobilization of intracellular calcium. EFs also modulated cytokine production selectively and can augment some effects of conventional polarizing stimuli on cytokine secretion.
Human T cells, which are present in wounded epithelia (skin or airway), also displayed directed migration toward the cathode and activation upon stimulation with physiological EFs (50 or 150 mV/mm). We showed for the first time that EF stimulation downregulated T cell activation following stimulation with antigen-activated APCs or anti-CD3/CD28 antibodies, as demonstrated by decreased IL-2 secretion and proliferation. Mechanistically, STAT3 modulation by the EF was implicated in the functional changes.
There are increasing efforts to use electrical stimulation to aid wound healing. This is especially important for recalcitrant or chronic wounds prone to infection, such as diabetic foot ulcers. Our work can inform design of electrical wound dressing materials seeking to target immune function and infection.

Biophotovoltaic (BPV) devices are a relatively new discipline in solar cells that utilize the components of natural photosynthetic proteins such as Photosystem I/II (PSI/II) from, for example, algae, plants, or cyanobacteria to effect photon-induced charge separation. The natural abundance and ease of isolation of these complexes, particularly PSI, make them ideal candidates for integration in biophotovoltaic devices. PSI converts photon energy into spatially separated electron/hole pairs. But does so with ∼ 100% internal quantum efficiency by effectively building a precise, nano-scale junction into each protein complex. Our first work describes the fabrication of microfluidic devices with a focus on controlling the orientation of Photosystem I complexes, which directly affects the performance of biophotovoltaic devices by maximizing the efficiency of the extraction of electron/hole pairs from the complexes. The surface chemistry of the electrode on which the complexes assemble plays a critical role in their orientation. Recreating that self-assembly-driven precision and mimicking the replacement of degraded protein are two (of many) major challenges to the design of stable, high-efficiency devices. We addressed both of those problems with a simple device design that allows the circulation of fresh PSI through devices that use linkers to direct the equilibrium of a self-assembly process towards oriented, active complexes. The basic design of the BPV devices used to investigate the phenyl-C61-butyric acid (PCBA) and a peptide (IQAc, sequence: IQAGKTEHLAPDC which a cysteine residue was attached to the C-terminus) linkers is a co-fabricated microfluidic channel that defines an electrode comprising the liquid metal eutectic Ga-In (EGaIn), which is in contact with the electrolytic medium. The floor of the microfluidic channel, which is perpendicular to the EGaIn electrode, supports a nanostructured gold electrode fabricated by shadow evaporation. The performance of devices fabricated using a fullerene, PCBA, was superior to that of a peptide, IQAc, as determined via the short circuit current, open circuit photovoltage, and power output. The PCBA linkers self-assemble onto nanopatterned gold electrodes, simultaneously directing the orientation of PSI and facilitating the collection of photogenerated electrons which show that fulleroids are well-known acceptor materials in organic photovoltaic devices, and facilitate the extraction of electrons from Photosystem I complexes without sacrificing control over the orientation of the complexes, highlighting this combination of traditional organic semiconductors with bio-molecules as a viable approach to co-opting natural photosynthetic systems for use in solar cells.
Also, to achieve continuous, optimal charge-transfer flux through multiple layers stacked on top of each other in solid-state BPV cells and to maximize their efficiency, the PSI complexes need to be kept properly aligned with respect to the hole/electron injection and structurally intact. In other work, we achieved a record efficiency of 0.64% for solid-state biophotovoltaic devices by tailoring the transport layers to maximize the extraction of charges from a layer of photosystem I. Specifically, we inserted complexes of gold nanoparticles into reduced graphene oxide to adjust the level alignment without sacrificing carrier mobility. We paired that layer with a polytyrosine-polyaniline hole-extraction layer. Our results demonstrate that the functionality of photosystem I in the non-natural environment of solid-state biophotovoltaic cells can be improved through the modification of electrodes with efficient charge-extraction layers. The decoration of graphene electrodes with gold nanoparticles is a generalizable approach for enhancing charge transport across interfaces, particularly when adjusting the levels of the active layer is not feasible, as is the case for photosystem I and other biological molecules.

Organic electrochemical based devices (OEDs) have been the main platform of many interesting developments in recent years. From highly sensitive biosensors all the way to intricate brain-inspired memories, OEDs have paved exciting new roads within Organic Electronics and beyond. Despite the unquestionable success of OEDs, the fundamental processes that govern their operations and sensing abilities, especially in reference to the ion-to-electron transduction is yet to be understood. This is translated to the lack of robust models to describe both the steady-state and transient characteristics of OEDs and electrochemical biosensors alike. Here, we present a thermodynamic-based model that quantitatively describes the operation of OEDs. We focus special attention to the description of Organic Electrochemical Transistor (OECT), given its importance within OEDs, and offer alternative interpretations to the “figure of merit” μC*, which is the fundamental materials component to the ion-electron transduction process. Finally, by analyzing a large amount of experimental data, with distinct active materials and electrolytes, we were able to come up with general guidelines for material design and device developments, towards highly sensitive electrochemical biosensors.

Molecular diagnostics, in particular nucleic acid testing, is critical in a wide range of fields relevant to the quality of human life, including a fundamental understanding of physiological processes, pathogen detection, and disease prognosis and diagnosis. Over the past few decades, quantitative polymerase chain reaction (qPCR) has remained as the gold-standard technique for nucleic acid testing. However, qPCR is limited by expensive instruments, the need for well-trained personnel operation, and long sample-to-results time. Recently, the discovery of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated) systems has opened a new path for nucleic acid detection. Leveraging the single-nucleotide target specificity of CRISPR-Cas systems as a result of complementarity between guide RNA and target nucleic acid sequence, scientists have developed a variety of CRISPR-based biosensing strategies for ultrasensitive and highly selective nucleic acid detection. However, the application of current CRISPR-based molecular diagnostics still requires lengthy and complicated target amplifications. In this talk, I will present our recent efforts on the development of a set of CRISPR-based molecular diagnostic platforms without the need for target pre-amplifications by coupling CRISPR assay with bioelectronics of graphene field-effect transistors and other electrochemical biosensors.

Long term monitoring of gastrointestinal function often requires hospital-based medical interventions which are generally limited to transient optical evaluation as well as short term monitoring of temperature, pH or impedance for generally hours and up to 48 hours. Long term monitoring of both the gastric and duodenal compartments stands to transform our capacity to monitor feeding activity as well as liver and pancreatic function. Here we report the development of a system which combines wireless communication with multi-mode sensing in the form of miniaturized flexible-board electronics with thin film sensors compatible with ingestion. The entire system can be administered either endoscopically or ingested and operate over an extended period. Once active, the device naturally conforms to the GI tract and has the capacity to transiently anchor itself at the level of the pylorus through which integrated sensors can be deployed to support the collection of electrophysiological signals. In vitro and in vivo evaluation support the device’s long-term stability as well as their applicability in measuring local temperature, pressure and impedance in GI tract that serve as important physiological indicators for GI diseases such as inflammation, gastroenteritis, mobility disorders, bloating, ulcers, intestinal ischemia, and bile reflux.

A urinary catheter is a flexible tube widely used when patients have difficulty urinating naturally and collect urine in a drainage bag. Patients prefer indwelling catheters to avoid multiple insertions of the catheters. However, in many cases, germs like bacteria can enter the body through the catheters and cause an infection in the bladder or kidney called a catheter-associated urinary tract infection (CAUTI). CAUTI affects 0.4 million people every year and causes patient distress, discomfort, pain, and activity restrictions and in some cases can lead to life-threatening conditions. Moreover, CAUTI incurs a medical burden of $340–450 million per year in the US. While many conventional tools such as invasive Cystoscopy, Non-invasive Ultrasound/CT scan have widely used been used for the detection of CAUTI, they are expensive, not real-time, and need manual intervention. A low-cost, non-invasive and contactless automatable sensor is critical in tackling CAUTI.

To address this need, here, we develop a non-invasive smart sticker platform that integrates low-cost smart stickers with a portable reader to detect early-stage UTI through continuous monitoring of urinary frequency and conductivity of urine. Inflammation of the bladder, urethra, or both causes a sensation of the need to urinate thereby increasing the urinary frequency above the healthy range of 5-8 times in 24 hours. UTI causes an increase in the bacterial count and biofilm formation in the flushed-out urine, effectively reducing the conductivity of the patient’s urine at the onset of infection. To capture the urinary frequency through level detection and bacterial infection through conductivity measurements, the smart stickers are designed as two parallel metallic strips manufactured using laser processing of metalized PET tape. This scalable manufacturing technique offers a platform for the rapid production of smart stickers using roll-to-roll processing. Using this platform, a long smart sticker of size 20 cm x 2 cm is manufactured for urine level monitoring to cover the urine bags of volume 2000 mL. Next, to achieve conductivity detection, a short sticker of size 4 cm x 2 cm is manufactured to ensure that the sticker is insensitive to level changes.

The achieve non-invasive measurements, the stickers are attached to the surface of the urine bag and interfaced with a portable reader with the help of a snap-on magnetic connector. The portable reader consists of a signal generator and a scalar reflectometer to form a portable network analyzer. When the urine level increases, the capacitance between the long stickers increases leading to a shift in the resonant frequency. When the conductivity of urine increases, the attenuation between the short stickers increases leading to a reduction in the amplitude of the resonant peak. The readings from the network analyzer are collected by an Arduino module and transmitted using a WiFi module.

A systematic characterization of the smart stickers is performed using artificial urine placed inside the urine bag. The level detector demonstrates a frequency shift from 530 MHz to 234 MHz when the urine level is changed from 500 mL to 2000 mL. Furthermore, the smart stickers provide constant potential voltage over 24 hours of measurement and show an excellent linear sensitivity of 3.1 mV/(mS/cm) from 10 mS/cm to 40 mS/cm in the urine sample. A proof-of-concept analysis is performed to simulate CAUTI by spiking the bacterial CFU in the urine and the readings demonstrated a 10% decrease in the conductivity when CFU levels surpassed the threshold of 10⁵ CFU/ml. The smart stickers presented in this work could be the first step towards the development of low-cost and automated noninvasive smart sensor tags for early-stage CAUTI detection.

In an increasingly health-aware population and a move towards remote diagnostics, remote healthcare monitoring, cloud-based med-tech, etc, we tap on the ubiquitous and unlimited reservoir of on-skin biomarkers, including sweat metabolites, in developing a wearable, non-invasive, continuous and real-time sensor – a printed, multiplexed biosensor device. In this talk, we highlight three components of our biosensor. Firstly, we look at the overall framework, of how the device is fabricated by a fully printed process, and what the integrated sensor is capable of measuring.[1-4] Carbon-based electrodes are promising candidates for developing cheap, miniaturized, and disposable biosensors for effective wearable applications. From developing our own printable carbon-based ink, to screen-printing the electrodes, to functionalising each electrode into a specific biosensor, and finally to multiplexing and integration; we show how these devices can potentially add value to home-based biomarker monitoring. Secondly, we look at a variation of our flexible electrode, a graphite/carbon fiber (G/CF) hybrid resembling a super-efficient 3D highway network where ample expressways (CF) run through numerous small factories (locally distributed graphite flakes) for rapid goods pick-up and transportation (electron transfer).[5] Lastly, we zoom in on the top-most layer of the device, the Kirigami paper sweat channel, critical in enabling real-time fresh-sweat reading.[6,7]

[1] A graphite-based paste/ink for electrochemical sensors, WP Goh, CY Jiang, Y Yu, XT Zheng, YX Liu, L Yang, PCT/SG2022/050407, Singapore (filed, secondary), 2022.
[2] XT Zheng, WP Goh, CY Jiang, Y Yu, YX Liu, L Yang et al, Manuscript in preparation.
[3] Stretchable biochemical interface for solid epidermal analytes, YX Liu, WP Goh, XT Zheng, Y Yu, SCL Tan, CY Jiang, RT Arwani, L Yang, SG-10202202318P, Singapore (filed, primary), 2021.
[4] RT Arwani, WP Goh, CY Jiang, XT Zheng, Y Yu, YX Liu, L Yang et al, Manuscript in preparation.
[5] Y Yu, L Yang et al, Materials Today Advances, 14, 100238 (2022).
[6] A kirigami paper fluidic channel for sweat sensors, CY Jiang, WP Goh, XT Zheng, Y Yu, YX Liu, L Yang, SG-10202113328Q, Singapore (filed), 2021.
[7] CY Jiang, WP Goh, SCL Tan, XT Zheng, Y Yu, YX Liu, L Yang, Manuscript in preparation.

Flexible, soft, self-healing, and adhesive conductive materials with Young’s modulus matching biological tissues (with a moduli of 0.5-500 KPa)¹ are highly desired for applications in bioelectronics. Here, we report self-healing, stretchable, highly adhesive and conductive films obtained by mixing tannic acid (PVA), ethylene glycol, and Poly (3,4-ethylenedioxythiophene) doped with polystyrene sulfonate (PEDOT:PSS).² Our films demonstrated remarkable self-healing and self-adhesiveness, compliance on skin, outstanding stretchability, high adhesion on skin , and low Young’s modulus. Our films were used to fabricate electrophysiological epidermal patch electrodes, which exhibited high-quality recording of electrocardiography (ECG) and electromyography (EMG) signals, like commercial Ag/AgCl gel electrodes.

Diabetic foot ulcers (DFUs) have affected millions of people in the U.S. and posed heavy burdens to patients and the healthcare system due to their being slow to heal, high recurrence rate, and potential risk of amputation and even premature death. A mainstay of DFU therapy is mechanical offloading to mitigate pressure at the ulcer, which, however, is usually not effective due to patients’ diminished sensation capabilities on the foot and low adherence to offloading recommendations. I will present our recent effort in developing a soft, flexible, wireless pressure-sensor-integrated smart bandage system for continuously monitoring the pressure on the foot and alerting the users to offload under exceeded pressure. The pressure sensor exhibits high linearity, negligible hysteresis, fast response time, and excellent water-proof properties relevant to this application. Wireless data transmission onto a smartphone via Bluetooth enables the display and interpretation of the measured pressure with the option of a preset pressure threshold, when exceeded, an alert will be generated for immediate notifications. Trials on healthy subjects demonstrate reliable monitoring performance and effectiveness in aiding offloading. The developed smart bandage system represents a significant advance in the prevention and management of DFUs and other pressure injuries as well as compression therapy corrections.

Wide-range light detection from the visible to the near-infrared (NIR) region is central to many applications such as high-speed digital cameras, autonomous vehicles, IR sensing, and wearable electronics. Recently, organic photodetectors (OPDs) were shown to be a promising platform for these applications. However, the conventional OPDs are often limited by low responsivity, narrow absorption range, large dark/noise current under applied bias, and large-scale production of these devices require halogenated processing solvents that have a negative environmental impact. In this talk, I will discuss strategies to reduce dark current in NIR OPDs based on a bulk heterojunction (BHJ) comprised of a polymer donor and a non-fullerene acceptor and the molecular design rule for processing OPDs from a green solvent, 2-methyltetrahydrofuran (2-MeTHF). Our OPDs exhibit an excellent specific detectivity (D*) over a broad wavelength range (400 -900 nm) and is comparable to those of commercial silicon-based photodiodes. We demonstrate that the PM7-D5:Y12 OPDs can be used as wearable self-powered devices to monitor heart rate and blood oxygen saturation.

Conventional magnetoelasticity, defined as the change in magnetic property in certain materials under mechanical stimulations, is usually observed in rigid alloys such as Tb_xDy_1-xFe₂ (Terfenol-D) and Ga_xFe_1-x (Galfenol). However, interfacing electronic devices based on these rigid alloys with the human body might hurt the surrounding tissues and the resulting scars will damage or even disable the devices. Herein, we discovered the giant magnetoelasticity in soft matter and achieved a magnetomechanical coupling factor of up to 6.77×10^-8 T/Pa without the need of an external magnetic field, which is up to five times larger than that of traditional rigid metal-based counterparts.¹ Meanwhile, the soft matter with the giant magnetoelasticity demonstrates a strain of up to 500% and a Young’s modulus as low as 166.2 kPa, which is well comparable to the human tissue and skin. To elucidate the fundamentals of giant magnetoelasticity in soft matter, we established an analytical model based on the magnetic particle and dipole interaction, which is well consistent with the experimental observation.
Then we explored our discovery in bioelectronics by coupling it with magnetic induction to invent a textile magnetoelastic generator (MEG) for biomechanical-to-electrical signal conversion. The human body is particularly rich in biomechanical energy,² which can be explored to power bioelectronics for in vivo and in vitro diagnostics and therapeutics. Intimately interfacing the textile MEG with the skin, the relevant body movements could deform the device and strongly alter its magnetic flux density, thereby inducing a current in the textile coil with a density of 1.37 mA/cm². The collected electricity in the capacitor could drive a commercial thermometer for personalized sensing.
Meanwhile, the textile MEG can work as a self-powered sensor for in situ biomechanical signal analysis.^1,3 Textile-based bioelectronics can realize wide biomedical applications while maintaining their initial wearing comfort.^4-8 Thus, we seamlessly sewed the textile MEGs on the chest area of clothes for respiratory monitoring.¹ With a fundamentally new working mechanism, the textile MEG demonstrated an intrinsic waterproof property, an sensitivity of 0.27 mA/kPa, a signal-to-noise ratio of 61.8 dB, and a response time of 15 ms, which could continuously monitor the strength, frequency, and patterns of various respiratory activities on heavy perspiration skin without any encapsulation. Assisted by a machine learning algorithm, respiration abnormalities, such as cough and rapid breathing, can be recognized with an accuracy of up to 90.89%, hence allowing a personalized and timely detection of respiratory diseases. Thus, we believe that the discovery of giant magnetoelasticity can branch out into broader soft-matter systems, illuminating the future of biomedical society by developing human-body-centered bioelectronics for personalized healthcare.
1. Chen, G.et al., Discovering Giant Magnetoelasticity in Soft Matter for Electronic Textiles. Matter 2021, 4, 3725-3740.
2. Chen, G.et al., Smart Textiles for Electricity Generation. Chem. Rev. 2020, 120, 3668-3720.
3. Zhao, X.*; Chen, G.*et al., Giant Magnetoelastic Effect Enabled Stretchable Sensor for Self-Powered Biomonitoring. ACS Nano 2022, 16, 6013-6022.
4. Chen, G.et al., Electronic Textiles for Wearable Point-of-Care Systems. Chem. Rev. 2022, 122, 3259-3291.
5. Chen, G.et al., Textiles for Learning Tactile Interactions. Nat. Electron. 2021, 4, 175-176.
6. Libanori, A.*; Chen, G.*et al., Smart Textiles for Personalized Healthcare. Nat. Electron. 2022, 5, 142-156.
7. Chen, G.et al., Textile Triboelectric Nanogenerators for Wearable Pulse Wave Monitoring. Trends Biotechnol. 2021, 39, 1078-1092.
8. Pan, H.*; Chen, G.*et al., Biodegradable Cotton Fiber-Based Piezoresistive Textiles for Wearable Biomonitoring. Biosens. Bioelectron. 2022, Accepted.
(*equal contribution)

As modern history progresses, the emergence of new viruses and the global outbreak of related diseases pose a serious threat to public healthcare issues. Recently, coronavirus disease 2019 (COVID-19) has become a global pandemic. Currently, the gold standard for COVID-19 diagnosis is quantitative reverse transcription-polymerase chain reactions (qRT-PCRs). However, it is time-consuming and requires laboratory-based hospitals, making the countries with poor infrastructures struggle to control the spread of virus.
We here present a method to formulate a thick (>1 μm) antifouling coating with macro- and nano-scale pores for electrochemical diagnostic sensors with ultrahigh sensitivity using an emulsion ink. Traditional antifouling coatings such as poly(ethylene glycol) (PEG) and bovine serum albumin (BSA) have several limitations in biomedical applications: they physically adsorb onto the sensing surfaces, which cannot ensure full blocking of the active surfaces and they may detach from the surface, 2) they hinder electron transfer in the electrode surfaces, reducing the electrochemical signal. Oil-in-water emulsion can be utilized to form the multiscale porous antifouling coating at any thickness desired and the highly porous nature of the material greatly increases its surface area available for bioreceptor presentation and interactions with analytes. We designed CRISPR-based sensors for detection of SARS-CoV-2 RNA using an electrochemical (EC) enzymatic sandwich detection assay, which allows us to detect the SARS-CoV-2 without electrode surface inactivation. It is also desirable as an antifouling coating as it can be rapidly applied using nozzle-assisted printing in a form that is highly sensitive and robust, plus functional nanomaterials can be embedded within the layer.

While there has been considerable recent interest in flexible and conducting polymer (CP)-based implants for interfacing with the brain, nearly all of these studies have focused on passive electrode arrays with low channel counts. Active integration of CP-based electronics for large-scale recordings, on the other hand, is limited, as the current library of active organic electronics is restricted to organic electrochemical transistors (OECTs). OECT gating is traditionally achieved over a common gate electrode and electrolyte, which limits the control of individual components in a large array. In addition, when OECTs are used for active switching operations, ion uptake of CPs poses a major issue for in vivo neural applications where the high ionic transients (~μA) result in the undesired activation of nearby neurons and lead to high ionic-electronic cross-talk with the adjacent OECTs. Therefore, there is a significant need for generation of alternative CP-based circuit components to generate complex organic circuitries capable of operation with internal ions to avoid interference with the measured media. In this work, we introduce a new class of organic semiconductors which can be operated by controlling the electrophoretic drift of the internally trapped cations in the channel to generate unidirectional conductivity with electrical output analogous to silicon-based diodes. We tune these organic diodes to work in complementary regime with the existing OECT configuration and demonstrate that this circuit architecture can be used to generate a switch matrix for electrode selection on the neural interface with minimal crosstalk between adjacent pixels. We further show that this topology enables the generation of ultra-thin (50-μm x 8-μm), flexible neural implants with high channel counts (32) that acquire local field potentials with high signal-to-noise ratio. To the best of our knowledge, this work is the first demonstration of active organic electrochemical circuits integrated in the front-end pixel of a fully implantable shank. Owing to their ionic-electronic conductivity, solution-based processability, and integrability with flexible, biocompatible substrates, unlike most inorganic circuit elements, CP-based circuitries are ideal to use on the neural interface.

A biosensor capable of continuously measuring specific molecules in vivo would provide a valuable window into patients’ health status and their response to therapeutics. Unfortunately, continuous, real-time molecular measurement is currently limited to a handful of analytes (i.e. glucose and oxygen) and these sensors cannot be generalized to measure other analytes. In this talk, we will present a biosensor technology that can be generalized to measure a wide range of biomolecules in living subjects. To achieve this, we develop synthetic antibodies (aptamers) that change its structure upon binding to its target analyte and produce an electrochemical current or emit light. Our real-time biosensor requires no exogenous reagents and can be readily reconfigured to measure different target analytes by exchanging the aptamer probes in a modular manner. Using our real-time biosensor, we demonstrate the first closed loop feedback control of drug concentration in live animals and discuss potential applications of this technology. Finally, we will discuss methods for generating the aptamer probes which are at the heart of this biosensor technology.

Osteoarthritis (OA) is a widespread disease which involves the degeneration of cartilage. Healthy cartilage derives its function from a combination of mechanical integrity of the collagen matrix and the physiochemical contributors of negatively charged aggrecan contained inside the matrix. While mechanical integrity via cartilage stiffness is a commonly used biomarker—primarily due to the wide availability of indentation tests—there are few approaches which focus on restoring the aggrecan content, which manifest as the swelling behavior of cartilage in response to different osmotic environments. The challenge is that cartilage explants—which have high physiological relevance—are too large for sensitive instruments and swelling changes are too little for simple visual or mass-based techniques. Here, we developed a platform which uses embedded graphene strain sensors to measure the swelling behavior of equine cartilage explants in real time. By simulating OA-like degradation through a series of enzymes, we were able to identify distinctive swelling profiles of explants undergoing proteoglycan digestion versus broad spectrum matrix damage. This parallel and scalable platform would enable new approaches to OA regeneration based on restoring proteoglycan content.

The following work focuses on the surface modification and processing of flexible PLA and PLA-Graphene composites using room temperature and confined micro plasma-based Townsend discharges in ambient conditions. The work includes in-situ-based processing using a proprietary plasma-integrated 3D printing system developed in the Energy Devices and Plasma Applications Laboratory at California State University, Fresno. The current-voltage characteristics of the plasma are varied based on the use of a quasi-static DC and a pulsed discharge with a variable voltage of 1 kV to 2.2 kV and with currents below 1mA. The micro plasma-based discharges integrated with the additive manufacturing process can tailor the surface characteristics of the composites by modification of their surface energies. The surface characterization of the 3D printed geometries is performed using profilometry, scanning electron microscopy, and electrical impedance spectroscopy. The surface energy and wettability characteristics are studied using contact angle characteristics. The micro plasma is characterized using high voltage probes, and visible spectroscopy. These 3D-printed geometries can be used as embedded wafers for targeted drug delivery.

Organic material-based conformable electronics are optimal candidates for components of bioelectronic circuits due to their inherent flexibility and soft nature. We have shown that internally ion-gated organic electrochemical transistors (IGTs) operate by leveraging ion reservoirs inside their channels to dramatically improve the operation speed and integration capacity of this ion driven transistor. In order to establish IGTs as versatile building blocks of bioelectronics, it is critical to establish fabrication strategies to enable IGT-based high-density integrated circuits for creation of fully implantable, wireless, and battery-free devices. Here, we report a scalable, self-contained, and integrable sub-micron internal ion-gated organic electrochemical transistor with vertical channel (v-IGT). Owing to the sub-micron channel size, v-IGTs can operate in the MHz range, surpassing any other electrochemical device while maintaining a high amplification. Furthermore, we analytically and experimentally derived the non-linear relationship between transistor speed and contact area as a function of gate and channel capacitance. The device is encapsulated using long-term implantable grade (parylene-C) material ensuring operational stability of over 1 year. We also developed a micron-scale perforation strategy to ensure intact hydration paths for transistor channels while maintaining effective isolation of densely packed adjacent transistors and minimizing potential for cross-talk and gate current leakage. The presence of this hydration via does not cause a detriment to the device characteristics due to the hydrophilicity and sufficient containment of ion reservoirs in the channel. We fabricated a conformable v-IGT-based array with 155k transistor/cm2, highlighting the viability and feasibility of IGTs as transistor architecture for a majority of flexible electronics applications. We further showcase their functionality by creating high-gain multi-stage amplifiers, oscillators, and multiplexers. Finally, we created a novel wireless, battery-free strategy for electrophysiological signal acquisition, processing and transmission that employs v-IGTs and ionic communication (IC). The wirelessly-powered v-IGTs were able to acquire and modulate neurophysiological data in-vivo and transmit them across the dermis, eliminating the need for any hard Si-based electronics in the implant.

The great advancement in nanopore technology has enabled real-time sequencing without the need for sample amplification with long read length. However, the ionic-current-based readout has limitations in the specificity and resolution, leading to higher error rate. The growing interest in analyzing other biomolecules such as proteins using nanopore configurations also demands integration of additional readout mechanisms such as recognition tunneling and metal-gap enhanced Raman. However so far there is very little success in the integration of single-molecule delivery, and multi-mode detection of the same molecule. We recently developed a new strategy to fabricate a single-molecule sensing microchip platform, Quantum Tunneling NanoElectroPore (Q-NEP), which integrates inside a nanofluidic channel a pair of tunneling electrodes self-aligned with a nanopore, by combining standard top-down lithography on standard semiconductor substrates with in situ reversible electrochemical deposition with feedback control. We have demonstrated simultaneous detection of DNA translocation events from coincidental ionic and tunneling current signals, and successfully obtained quantized conductance from short thiolated oligonucleotides bridging the tunneling gap as they were delivered through the nanopore. In addition, metal-gap enhanced Raman signals are also obtained from L-cysteine molecules through the device. These results provide the first step for the integration of ionic, tunneling current and Raman multi-mode analysis of individual molecules on the Q-NEP platform, and reveal the promising potential for a unified microchip platform for multi-omics analysis with high resolution and specificity.

A large millimeter-wide electronic interface can detect at a single-molecule/entity limit-of-detection. The technology is called SiMoT - Single-Molecule with a large Transistor.¹ So far, antigens (Immunoglobulins, C-reactive proteins, spike 1, HIV p-24), antibodies (anti-immunoglobulins, anti-spike1), peptides, viruses (SARS-Cov-2), bacteria (Xylella fastidiosa), and even DNA strands (KRAS, miR-182) have been detected. Selectivity is assured by covering the gate electrode with a large number (10¹¹-10¹²/cm²) of recognition elements to affinity binding the target element.

SiMoT detects directly in a droplet (0.1 mL) of a real fluid such as saliva from COVID-19 patients, blood serum, pancreatic cysts juice, and olive saps from infected trees. Relevantly Brownian diffusion enables the entity to statistically hit the millimeter-wide interface in a few minutes.²Considering the footprint of a molecule on a millimeter-wide interface, it is like spotting a droplet of water falling on the surface of a 1 Km wide lake as depicted in the graphical abstract.

The applications span from a handheld intelligent single-molecule binary bioelectronic system for fast and reliable immunometric point-of-care testing of COVID-19 patients³ and Xylella fastidiosa single bacterium detected in infected plants sap. The phenomenon enabling such outstanding performance level was discovered in 2018.⁴ While still under investigation, it is supposed to involve an amplification that starts from the single affinity binding that triggers a propagating collaborative response.

Future actions include the deepening of our understanding of the sensing mechanism and the engagement in a campaign of thousands of clinical trials that will bring SiMoT beyond TRL5.

References
1. E. Macchia et al. Chemical Review 2022, 122, 4636 DOI: 10.1021/acs.chemrev.1c00290
2. E. Macchia et al. Advanced Science 2022, 2104381 DOI: DOI: 10.1002/advs.2021043811
3. E. Macchia et al., Science Advances 2022, 8 (27) DOI: 10.1126/sciadv.abo0881
4. E. Macchia et al., Nature Communication 2018, DOI: 10.1038/s41467-018-05235-z

Pulse oximetry is the most widespread method of monitoring patient heart rate and blood oxygen levels in both clinical and non-clinical settings. These devices tend to be ridged, bulky, and therefore suitable only for short-term use. In addition, they suffer degraded performance due to movement and changes in ambient light levels, and can present variability from one individual to another. To facilitate long-term monitoring of physiological parameters, including heart rate and oxygen saturation, implanted devices are required. Implantation removes interference from external light sources, eliminates sensitivity to skin pigmentation, and allows enhanced performance during movement associated with greater sensitivity.
Here we demonstrate a low cost, ultra-flexible pulse oximetry probe. The hybrid flexible devices are fabricated on 5 µm thick Parylene C using laser ablation to define the circuit design, and integrate a small ridged photodetector and LEDs using a flexible adhesive. Finally, contact between the probe and the processing board is achieved using a flat flexible cable and anisotropic conductive film. Devices are demonstrated in vivo on a sedated porcine subject and calibrated against a standard peripheral SpO₂ meter while the subject’s oxygen intake was varied. In addition to demonstrating the functionality of these implanted devices, we show that the implementation directly on the femoral artery of our devices records a more acute response to the variation in oxygen intake compared to the peripheral measurements.
These devices are low cost and based on biocompatible materials, and can be easily implanted during cardiovascular surgery. This offers a route towards long-term implantation of devices for continuous patient monitoring.

In this presentation, we introduce a skin-compatible meta-structured e-skin patch that only consists of commercial adhesive dressing and FPCB for continuously collecting electrocardiographic signals over five days. In terms of long-term monitoring, it is crucial for the on-skin sensors to stably monitor bio-signals without any skin discomfort. Our approach presents a practical and cost-effective way to manufacture a skin-compatible wearable sensor system, making use of conventional materials that were difficult to include due to their high elastic modulus.
To rationally design the metal structural pattern of a stretchable sensor patch suitable for skin movement, we measured and analyzed the deformation field around the chest by using digital image correlation method and finite element simulation. Among many possible patterns, we chose the Kagome-based structure for the adhesive dressing that can well accommodate the deformation of the skin as well as give breathability to the patch due to its high porosity. We further devise a tethering scheme of attaching the FPCB to the adhesive dressing. The results showed that our sensor patch system greatly improved skin comfort and was able to monitor ECG signals for five days without any discomfort.

The rather reactive and centralized medical approaches in today’s healthcare system prevent individuals from obtaining diagnosis and treatment in a timely and accessible manner. Presently, commercially available devices for remote health monitoring are majorly limited to body’s vitals and glucose levels, limiting access to healthcare. In this talk, I will present the wearable biosensors which utilized sweat to extract individual’s biomolecular information. These sensors integrate flexible electronics and solid-state electrochemical sensors in a single robust platform to achieve reliable analyte quantifications at the site of sweat collection. Specifically, the wearable ultralow-volume sweat sensors make sweat a viable mode of health monitoring at the molecular level across activities, whether active or sedentary, and across user groups, whether young or old, healthy or ill. By using the sensors, continuous sweat analysis can be used to study how the body’s endogenous and stimulated sweating response relates to stress, metabolic conditions, and potentially neurological afflictions through multiplexed quantification of ions, metabolites, and drugs.

Printed carbon based electrolyte-gated transistors represent ideal ionic/electronic hybrid devices that bridge biology and electronics. These devices are being investigated for a wide range of applications, from drug delivery to neuromodulation and highly sensitive biosensing. Here we report our recent progress in printed devices based both on thin films of polymer semiconductors and randon-networks of carbon-nanotubes. In particular, we show how large-area printed arrays of electrolyte-gated polymer transistors can enable future diagnostic tools to target specific biomarkers at clinical level. At the same time we propose printed carbon-nanotubes as an alternative approach for highly stable biosensors operating in acqueous environment. Besides their use to target biomarkers, we also show how such printed carbon-based transistors can be employed to monitor cell cultures proliferation over consecutive days. Remarkably, we also demonstrate that, despite the planar geometry of the device, spontaneous recording of intracellular action potentials of cardiomyocytes can be detected. Such results show that printed carbon-based electrolyte-gated transistors have great potentials for many aspects of future large-area bioelectronics, including diagnostic and parallel probing of electrogenic cell cultures.

My team’s efforts are focused on the development and engineering of nanomaterials-based flexible platforms to interrogate and affect the properties of tissue, with a specific goal to understand signal transduction (chemical or electrical) in tissue or complex 3D cellular assemblies. Highly flexible bottom-up nanomaterials synthesis capabilities allow us to form unique hybrid-nanomaterials that can be used in various input/output bioelectrical interfaces, i.e., bioelectrical platforms for chemical and physical sensing and actuation. We developed a breakthrough bioelectrical interface, a 3D self-rolled biosensor arrays (3D-SR-BAs) of either active field effect transistors or passive microelectrodes to measure both cardiac and neural spheroids electrophysiology in 3D. This approach enables electrophysiological investigation and monitoring of the complex signal transduction in 3D cellular assemblies toward an organ-on-an-electronic-chip (organ-on-e-chip) platform for tissue maturation investigations and development of drugs for disease treatment. Utilizing graphene, a two-dimensional (2D) atomically thin carbon allotrope, we can simultaneously record the intracellular electrical activity of multiple excitable cells with ultra-microelectrodes that can be as small as an axon (ca. 2µm). The outstanding electrochemical properties of the synthesized hybrid-nanomaterials allow us to develop highly efficient catalysts, and electrical sensors and actuators. We demonstrated sensors capable of exploring brain chemistry and sensors/actuators that are deployed in a large volumetric muscle loss animal model. Finally, using the unique optical properties of nanocarbons in the form of graphene-based hybrid-nanomaterials and 2D nanocarbides (MXene), we have formed remote, non-genetic bioelectrical interfaces with excitable cells and modulated cellular and network activity with low needed energy and high precision. In summary, the exceptional synthetic control and flexible assembly of nanomaterials provide powerful tools for fundamental studies and applications in life science and potentially seamlessly merge nanomaterials-based platforms with cells, fusing nonliving and living systems together.

Low-cost, flexible, and lightweight electronic biosensors offer an exciting pathway to future products for environmental and plant health monitoring. Organic electrochemical transistors (OECTs) are particularly favorable sensing platforms due to their high sensitivity, low power requirements, and use of soft, biocompatible materials. Furthermore, OECTs can be manufactured with scalable printing techniques, which makes them suitable for large-area electronics (LAE) applications. This presentation will describe the development of fully printed, flexible OECTs that can accurately detect a suite of analytes (e.g., nutrients, pH, temperature) that are relevant to plant health. For example, the devices were used to accurately detect macronutrient concentrations in whole plant sap and hydroponic fluids in real-time. The rheological and electronic properties of the OECTs were tuned by doping the semiconducting channel with sugar alcohols, super absorbing polymers and thickening agents. These additives boosted the transconductance and sensitivity of the transistors, which led to a super-Nernstian response to the target analyte. By utilizing ion selective membranes (ISMs), we fabricated OECT-based ion sensors that demonstrate selectivity to the target ion against similar ions over five orders of magnitude in concentration and a limit of detection (LOD) as low as 10 µM. The results confirm that lightweight, printed organic electronics are a suitable path to high-throughput, low-cost assessment of plant health, which could lead to massive efficiency gains in agriculture.

With the emphasis of healthcare shifting towards prevention and early detection of diseases and monitoring of chronic conditions, there is a growing need for hassle-free telemedicine sensor technologies that can be seamlessly integrated into daily life. While significant progress has been made in the development of wearable sweat and salivary biosensors to meet this need for rapid, real-time collection of physiological information, the majority of current epidermal sensing systems are unable to detect trace-level disease-relevant biomarkers accurately in biofluids and cannot be mass produced. To meet this demand for low-cost, mass-producible mHealth devices for at-home settings, we developed several fully-integrated laser-engraved graphene-based biosensors for the detection of low-concentration sweat and salivary analytes including hormones (e.g. cortisol) and proteins (e.g. SARS-CoV-2 nucleocapsid protein and C-reactive protein). Several graphene surface engineering strategies are investigated for the sensitive and selective detection of targets. System-level engineering and microfluidic designs are explored to achieve on-demand sweat induction and harvesting under sedentary settings, automated sweat and reagent routing, and in situ signal correction and analysis for facile operation on the skin. The utility of these fully integrated flexible mHealth systems is evaluated through multiple human studies involving healthy and various patient subgroups towards stress assessment, rapid COVID-19 diagnosis and severity assessment, as well as the monitoring and management of various chronic conditions including chronic obstructive pulmonary disease, heart failure, and inflammatory bowel diseases. These fully integrated mHealth devices demonstrate an enabling technology that can be easily adapted to monitor a broad spectrum of disease-specific proteins, cytokines, and hormones, advancing future applications in personalized disease diagnosis, management, and prevention.

Wearable biosensors represent a promising opportunity to monitor human physiology through dynamic measurements of (bio)chemical markers in bio-fluids such as sweat, tears, saliva, and interstitial fluid in a continuous and non-invasive way. Such new platforms can thus offer real-time (bio)chemical information toward a more comprehensive view of a wearer’s health, performance, or stress at the molecular level in daily-life. Continuous biomonitoring addresses the limitations of traditional invasive blood testing and provides the opportunity for early diagnostic and therapeutic interventions. My research is focused on developing wearable electrochemical biosensors towards non-invasive health monitoring opportunities and evaluating the potential impact of such wearable point-of-care devices on our daily life. The innovative biosensing technology was demonstrated by successful electrical/electrochemical signal transduction of biomolecular/biocatalytic reactions. It was further developed into various wearable form factors using printing technology, offering wearer conformity, flexibility, and stretchability. The wearable biosensors could measure a broad spectrum of biomarkers such as metabolites, electrolytes, hormones, and various small molecules in non-invasive biofluids, demonstrating clinical values through human studies. Furthermore, our recent outcomes will be discussed in this talk covering the discovery of new biomarkers and their underlying mechanisms utilizing various wearable form factors, such as mouthguards, contact lenses, and epidermal patches.

The development of micro-electronic devices/chips that bridge the gap between the rigidity of traditional electronics with the soft mechanics of biological systems is highly desirable. The emergence of highly conjugated polymers in combination with 2D materials has opened up exciting directions in biomedical research including point-of-care diagnostics. With the ultimate goal of fully integrated wearable sensors combined with IoT, and that of autonomous at-home diagnostic tests, organic/hybrid bioelectronic technologies have been heavily explored the past decade resulting in novel device configurations. This talk will summarize our recent efforts on developing biosensors based on organic electronic and 2D materials for sensing vital biomarkers, on rigid and flexible substrates showcasing the potential of novel materials and combinations thereof towards next generation point of care sensors.

Wearable devices are becoming ubiquitous. They are used extensively by consumers and are becoming more noticeable in the medical domain. But contemporary wearable devices are still relatively bulky and often have limited equivalence to medical gold standards. Skin patches with chemical, mechanical, and electrophysiological sensors offer new opportunities for future sensors. These devices can be designed to seamlessly interact with the user while also providing high quality and long-term data. In combination with miniaturized low power electronics and powerful data analysis algorithms these devices have the potential to provide continuous measures of various physiological parameters from freely behaving humans. In this presentation I will discuss some of our recent achievements in monitoring the electrophysiology (EEG, EOG, EMG and ECG) of freely behaving humans during rest, overnight sleep and during movement. I will also present recent progress towards simultaneous measurements of electrophysiology and blood pulsation at the arm, neck, and face.

Biocompatible electronics that avoid any damage to surrounding tissues are a substantial aspect of biomedical systems and wearable technology. The integration of electronic devices that interact closely with users, however, requires a high level of adaptability to surfaces and deformations during use, making stretchable electronics indispensable. Moreover, the rapid increase in the number of devices and their applications has raised significant environmental concerns, such as improper disposal of tech waste. Achieving a sustainable, technologically advanced future will therefore require tackling the e-waste problem. New concepts for degradable power sources that withstand the high-power requirements of modern electronic devices offer a pathway through their environmental friendliness. We introduce a new approach for fabricating high-power stretchable and biodegradable batteries as sustainable power sources. Here, a concept for merging intrinsically stretchable materials with engineered stretchability through component-level kirigami patterning to yield high-power biodegradable batteries with reversible elasticity of up to 35% for uniaxial strain and 20% for biaxial strain is demonstrated. Using a combination of molybdenum metal foils, a molybdenum trioxide paste, and magnesium metal foils as electrode materials, a peak power output of 196 µW cm^–2 and an energy density of 1.72 mWh cm^–2 is achieved. The biodegradable batteries are used to power a biomedical sensor patch on the skin that allows monitoring of sodium levels in sweat. This concept provides a versatile route for high-power biodegradable batteries, enabling untethered soft electronic devices in a sustainable future.

Materials and fabrication concepts for the creation of soft electronics coupled with miniaturization of wireless energy harvesting schemes enable the construction of high-performance electronic and optoelectronic systems with sizes, shapes and physical properties matched its biological host^[1]. Applications range from continuous monitors for health diagnosis to minimally invasive exploratory tools for neuroscience^[2]. Translation of these approaches towards neuroscience tools to enable advanced insight into the central and peripheral nervous require means to enable full subdermal implantation and operation to enable naturalistic behavior which often is the readout of circuit level behavioral experiments that are critical to decipher function. This talk presents science and engineering approaches for the creation of soft devices with near field power transfer and data communication capabilities^[3] and discusses application in devices for the stimulation and recording of organ systems such as the brain, the peripheral nervous, cardiovascular and the musculoskeletal system. We introduce a series of advances in subdermally implantable device technologies that enable digitally controllable subdermal platforms with multimodal optogenetic and electrical stimulation capabilities^[4][5] with the ability to provide stimulus without the physical penetration of the blood brain barrier. Additional to these stimulation capabilities we demonstrate new capabilities in biointerfaces with the cardiovascular and musculoskeletal system^[6] that enable recording of organ health as well as closed loop operation chronically in freely moving subjects. Because of the minimally invasive nature of these tools we show chronic neuromodulation capabilities over months in freely moving subjects enabling a digital disease models as well as platforms that can be rapidly translated to large animal models.
[1] T. Stuart, L. Cai, A. Burton, P. Gutruf, Biosens. Bioelectron. 2021, 113007.
[2] L. Cai, P. Gutruf, J. Neural Eng. 2021, 18, 41001.
[3] P. Gutruf, V. Krishnamurthi, A. Vázquez-Guardado, Z. Xie, A. Banks, C.-J. Su, Y. Xu, C. R. Haney, E. A. Waters, I. Kandela, S. R. Krishnan, T. Ray, J. P. Leshock, Y. Huang, D. Chanda, J. A. Rogers, Nat. Electron. 2018, 1, 652.
[4] A. Burton, S. M. Won, A. K. Sohrabi, T. Stuart, A. Amirhossein, J. U. Kim, Y. Park, A. Gabros, J. A. Rogers, F. Vitale, A. G. Richardson, P. Gutruf, Microsystems Nanoeng. 2021, 7, 62.
[5] J. Ausra, S. J. Munger, A. Azami, A. Burton, R. Peralta, J. E. Miller, P. Gutruf, Nat. Commun. 2021, 12, 1968.
[6] L. Cai, A. Burton, D. A. Gonzales, K. A. Kasper, A. Azami, R. Peralta, M. Johnson, J. A. Bakall, E. Barron Villalobos, E. C. Ross, J. A. Szivek, D. S. Margolis, P. Gutruf, Nat. Commun. 2021, 12, 6707.
[7] A. Burton, S. N. Obaid, A. Vázquez-Guardado, M. B. Schmit, T. Stuart, L. Cai, Z. Chen, I. Kandela, C. R. Haney, E. A. Waters, H. Cai, J. A. Rogers, L. Lu, P. Gutruf, Proc. Natl. Acad. Sci. 2020, 201920073.

Paper-based devices have recently emerged as a simple and low-cost paradigm for flexible and wearable applications. Paper is a flexible and low-cost game-changing substrate for next-generation electronics because of its excellent mechanical and dielectric properties with chemical and thermal stability. The biodegradability of paper-based electronics has attracted much attention as the future of green electronics, reducing the dramatic increase in electronic waste. Concurrent with advances in paper-based electronics, remarkable efforts have been dedicated to paper-based microfluidics for next-generation point-of-care (POC) diagnostics, particularly in limited-resource and remote regions. Fluidic components, patterned in paper, allow for instrument-free liquid transport via capillary wicking and storage of biological and chemical reagents within their 3-D fiber network of paper. A merged system incorporating paper-based electronics and paper-based microfluidics will have the transformative potential to yield exceptionally powerful functions and performances. In this invited talk, he will present many innovative paper-based electronics and paper-based microfluidics that his research group recently developed especially for flexible and wearable applications. Details of the frontier of research to improve the performance of the devices will be discussed, followed by a critical perspective on strategic future directions.

Understanding how neural activity evolves in a developing brain is of great scientific value and clinical importance. However, chronic in-vivo monitoring of neural activity in a fetal animal requires a minimally invasive, conformable, and miniaturized device. Here, we introduce a fully conformable, battery-free, implantable bioelectronics device integrating a neural probe, internal gated ion transistor(IGT), and ionic communication circuit(IC). We hypothesize that IGT could be wirelessly powered by a pair of IC electrodes sensing externally generated electrical potential energy within polarizable media, and IGT enables modulation of the IC current by electrophysiological signal that can be sensed externally. To validate this hypothesis, firstly, we have characterized IGT's performance under IC operation frequency. IGT shows no transconductance decrease when powered with 1MHz sine wave, compared with DC power. We have characterized the power efficiency and signal-to-noise ratio of this system. We demonstrated IC could effectively transmit mill-watt power to an implantable device and IGT could effectively modulate IC current with sub-millivolt gate signal. Finally, we fabricated a fully conformable, implantable device and implanted the device in baby mice. We demonstrated chronic recording of high-quality electrophysiological signals, including local field and action potentials over 30 days. In this work, we have demonstrated a fully conformable neural implant that establishes a safe, long-term pathway to the signal inside biological tissues.

Understanding the chemical signals governing plant physiology is critical for environmental monitoring and protection. Electrostimulation of electrical networks in plants can be linked to ion transport and the activation of ion channels, and can trigger biological activities such as gene expression or plant movements. Electrical currents have also been used to enhance plant growth. Here, we present a new technique to fabricate biocompatible adhesive sensors that can be placed as wearable dressing, and stimulate or relay signals emitted by the plant. The customizable sensors are used to induce electrostimulation and can be controlled wirelessly for over 1 week. This work represents a stepping stone toward understanding the biological mechanisms of plants, and evaluating their defense response when subject to stress.

The development of soft neural interfaces opens opportunities for long-term studying and modulating brain functions and diseases with fewer side effects by minimizing the mechanical mismatch between artificial devices and soft tissues. However, few designs have enabled both electrical and chemical sensing – necessary for capturing the diverse neural signals in vivo – as simultaneous high conductivity and redox surfaces are required for such multifunctional electrodes. Here we report a novel method leveraging the unique combinations of electrical conductivity, functional surfaces, and solution processibility of MXenes, an emerging class of 2D nanomaterials, to produce a thin conformal MXene coating on nylon filaments (30-300 µm in diameter) at a fast speed (up to 15 mm/s). The highly aligned MXene coatings provide the microelectrodes with an electrical impedance as low as 3Ω at 1kHz. In addition, the MXene coating provides ample redox surfaces for the detection of neural transmitters such as dopamine and 5-hydroxytryptamine. These MXene filament microelectrodes offer a robust, miniaturized platform for the monitor and stimulation of neural activities at the cellular level, facilitating a greater understanding of health and disease.

Wearable and flexible biosensors for the continuous monitoring of metabolites in sweat can detect a few analytes at sufficiently high concentrations, typically during vigorous exercise so as to generate sufficient quantity of the biofluid. I will introduce the design and performance of a wearable electrochemical biosensor platform for in situ analysis, in sweat during physical exercise and at rest, of trace levels of multiple metabolites, nutrients, and proteins. The biosensor consists of laser-engraved graphene electrodes that are functionalized with target-specific natural or synthetic receptors, and integrated with modules for iontophoresis-based sweat induction, microfluidic sweat sampling, signal processing and calibration, and wireless communication. The clinical value of our wearable systems is evaluated through human studies toward metabolic/nutritional assessment and disease management. These wearable technologies could open the door to a wide range of personalized monitoring, diagnostic, and therapeutic applications.

Laser-induced graphene (LIG) has established itself as a very attractive material for electrode fabrication, within several applications in bioelectronics. The straightforward, high throughput graphitization of several precursor materials using this laser conversion process allows for the simultaneous synthesis and patterning of this 3D graphitic material with diverse electrode architectures, to target several biosensing applications, from biophysical to biochemical monitoring.
Recently, paper has appeared has a viable alternative to conventional petroleum-based plastic polymer precursors, such as polyimide. This is due to the possibility to photothermally convert aliphatic cellulose monomers into graphene lattices, using several cellulose substrate treatment strategies. Application of fire-retardant chemical modifications and external aromatic moieties improves the graphitization potential of cellulose, to reach LIG films with 5 ohm.sq^-1 sheet resistance and conductivities as high as 67 S.cm^-1.
With these improved conductive properties of paper derived LIG, this precursor material can be easily employed for the fabrication of disposable biosensing units, where paper acts as both the support substrate and precursor material for conductive electrodes fabrication, without the need for more intricate printing techniques. Alternatively, strategies can be employed to separate converted and unconverted phases, through transfer methods, to make this material compatible with wearable applications.
In this presentation, we report the use of cellulose as a material in the toolbox of LIG precursors, aimed at the development of both disposable and wearable biosensing applications. Paper-based electrochemical sensors using this material are presented, aiming at disposable sensor development for different analytes. Glucose and pH electrochemical sensors were fabricated, showing the compatibility of the material with several sensing strategies, such as enzymatic and non-enzymatic sensing. To translate patterned electrodes for wearable applications, a straightforward transfer method is presented, using a water-induced peel-off method. This method is capable of efficiently separating unconverted cellulose and converted LIG phases, allowing for the transfer of LIG patterns onto flexible, conformable and elastomeric substrates with adhesive properties, for example medical grade adhesives. Using this method, electrochemical biosensors, strain sensors for biophysical monitoring and electrodes for electrophysiological signal monitoring are presented.
In conclusion, the concepts explored herein show the applicability of LIG towards the fabrication of robust point-of-care, disposable analytical devices, but also the its potential for integration in wearable sensing systems, aiming at more sustainable, accessible bioelectronic applications.

Neuron-on-a-Chip technology has been intensively studied in medical and pharmaceutical fields to understand and explore complex neurological systems in a controlled in vitro platform. This platform provides a biocompatible cell culture dish with multiple electrodes that can transmit bioelectrical signals and well for nourishing, guiding, and proliferating the neurons. Recently, many researchers studied and investigated three-dimensionally cultured organoids derived from human induced pluripotent stem cells (hiPSC) in in vitro systems. The organoids showed much similar behavior to in vivo systems than 2D cultured cells due to their capability of mimicking organ cell formation with cell-to-cell interaction. However, due to their self-organizing characteristics while development and proliferation, the design of the multielectrode array (MEA) and microfluidic channels should be considered an important factor in realizing practical preclinical models.
In this paper, we developed a MEA chip consisting of a total of 130 electrodes that each can act as an electrically stimulating or recording site and dumbbell-shaped microfluidic channel to investigate hiPSC-derived motor nerve organoids placed in two wells. To minimize the contact impedance of the electrodes, the opening area (9 um diameter) is covered with platinum black. The organoids were cultured for one month in vitro and organoid formation and axon growth were investigated. The results showed that the axons from the organoids extended ~3 times longer in narrow fluidic channel (300 um width) than inside the well (2 mm diameter). Developed MEA chip showed simultaneously detection of multiple neuronal activities with a low background noise level. Also, from the electrical stimulation tests, neuronal network activities including conduction behavior were measured and analyzed. Further improvements to the MEA chip including 3D shaped electrode array and microfluidic channel variation may provide a basis for an advanced preclinical model platform for regenerative medicine and human disease modeling in the future.

As the aging of society proceeds and the healthcare market has been significantly growing, it is essential to develop a musculoskeletal biological signal monitoring platform. Although most semiconductors or MEMS sensors for acquiring biosignals have excellent sensitivity and accuracy, they have the limitation of form factor for long-term attachment to human bodies [1]. In this study, we demonstrated a formfactor-free musculoskeletal platform integrating four types of sensors on a stretchable substrate that is stretched up to 20 % through an in-situ process. The stretchable substrate fabricated with serpentine electrode structure showed almost no change in the resistance of the electrode when it was increased up to 20 % strain, a highly reliable biological signal can be eventually obtained. Also, the sensors of Mechanomyography (MMG), Forcemyography (FMG), Electromyography (EMG), and accelerometer are integrated with one stretchable substrate, and the correlation between the sensors is analyzed to monitor the biological activity with high accuracy. In particular, MMG sensors are required high sensitivity and SNR to detect extremely low amplitude vibration signals inside the skin. Most MMG sensors are used ceramic-based microphones of accelerometers which are highly sensitive and accurate. However, it is not conformally adhered to human skin due to the rigid type of ceramic-based devices, thereby there is a constraint of data reliability caused by motion artifacts. To enhance the reliability of data, a stretchable 10-um-thick MMG patch sensor was fabricated by screen printing based on flexible 0-3 nanocomposite piezoelectric materials. Moreover, the SNR of the signal was improved by more than 3.5 times with the bump structure to the sensor. The pressure sensitivity was 395 mV/N (0%) and 407 mV/N (20%), respectively, before and after a stretch of 20 %, which confirmed negligible hysteresis with a delta value (Δ< 3). The MMG sensor can detect dynamic vibrations in the working frequency range from 1 Hz to 100 Hz. Finally, it was applied to recognize or predict motion intention by analyzing the correlation of biological activity through the four types of sensor signals acquired by isometric contraction and relaxation of flexor digitorum longus.
Acknowledgement
This work was supported by the Technology Innovation Program (RS-2022-00154781), funded By the Ministry of Trade, Industry & Energy (MOTIE, Korea) and Institute for Information & communications Technology Promotion (IITP) grant funded by the Korea government (MSIT) (Grant No.2022-0-0025).
Reference
[1] Scientific Reports volume 11, 2631 (2021)
[2] Scientific Reports volume 9, 5569 (2019)

Implantable neural probes that are inserted into the brain tissues are widely used in brain-machine interfaces (BMIs) for the analysis of brain circuits. They are implanted into specific brain regions such as Thalamus and Cerebellum, that generate neural signals related to electrophysiological information. Recently, flexible neural probes are developed to minimize the damage to brain tissues and nerves for reducing foreign body responses (FBRs) in the brain. To prevent FBRs between the probes and brain tissues, mechanical properties such as bending stiffness and Young’s modulus of devices should be well matched with that of the brain. However, conventional flexible neural probes using a silicon substrate still induce damage in the brain due to their even high Young’s modulus (> 1 MPa) than that of the brain (~ 1.8 kPa). Furthermore, the electrical properties of the devices such as conductivity and impedance could be deteriorated by focusing on only matching mechanical properties between the probes and the brain. Herein, we fabricated Au nanoparticles (AuNPs) embedded fiber-type neural probes with balanced mechanical and electrical properties for a long-term measuring brain signals. The fiber-type neural probe was fabricated by controlling the distribution of Au ions in polymeric networks to using osmotic pressure. After the Au ions were converted into AuNPs by the reducing agent, A core of the fiber-type neural probe was still remained as an original elastomer, and the AuNPs were formed on the exterior of the fiber. The proposed fiber showed remarkable electrical properties, including high conductivity (7.68 × 10⁴ S/m) and low impedance (2.88 × 10³ Ω at 1 kHz). In addition, the fiber exhibited brain tissue-like mechanical properties such as low Young’s modulus (170 kPa) and low bending stiffness (22.2 N m^-1). We could successfully measure various electrophysiological signals from a mouse brain using a brain chip containing the developed fiber-type neural probe. Additionally, the fiber-type neural probe exhibited significantly reliable signal recording with an impedance of under 4 kΩ at 1 kHz over four months post-implantation with negligible FBRs in the brain. We suggest that the developed fiber-type neural probe could be a crucial role in the field of neurological sciences and BMIs for understanding the mechanism of brain circuits and developing electronic devices in the treatment of chronic diseases.

Wearable chemical biosensors detecting low concentration of biomarkers which can indicate health disorders such as asthma, cystic fibrosis, and bronchiectasis have attracted considerable attention in the field of bioelectronics [1]. To improve the sensing performance, various materials including nanostructured metal-oxide semiconductors and low-dimensional nanomaterials have been extensively investigated as promising active sensing channels owing to their high reproducibility and large surface-to-volume ratio [2]. However, the chemical biosensors based on these materials inevitably require high operating temperatures and complicated fabrication processes, which can limit the applicability of wearable electronics in the systel level [3]. Recently, polymerized ionogels exhibit great potential in ionotronic applications owing to their high chemical stability, mechanical flexibility, and ionic conductivity in addition to molecular interactions [4]. In this presentation, we will demonstrate polymerized ionogels consisting of ionic liquids (ILs) incorporated with 3 dimensional polymeric neworks which immobilize the ILs while maintaining distinctive their properties. We will also demonstrate chemiresistive-type wearable chemical biosensors based on polymerized ionogels, which can detect biomarkers of nitrogen oxide molecules in exhaled breath down to ppb-level and exhibit excellent operational stability under various environmental and mechanical conditions such as humidity, temperature, and physical deformation. We believe that this work can stimulate the field of ionotronic biosensors based on polymerized ionogels for human breath analysis underlying biomarkers.

References
[1] C. Sánchez-Vicente, J. P. Santos, J. Lozano, I. Sayago, J. L. Sanjunrjo, A. Azabal, S. Ruiz-Valdepeñas, Sensors 20, 7223 (2020).
[2] A. Afzal, N. Cioffi, L. Sabbatini, L. Torsi, Sensors Actuators, B Chem. 171-172, 25-42 (2012).
[3] X. Zou, J. Wang, X. Liu, C. Wang, Y. Jiang, Y. Wang, X. Xiao, J. C. Ho, J. Li, C. Jiang, Y. Fang, W. Liu, L. Liao, Nano Lett. 13, 3287-3292 (2013).
[4] L. Chen, J. Fu, Q. Lu, L. Shi, M. Li, L. Dong, Y. Xu, R. Jia, Chem. Eng. J. 378, 122245 (2019).

Acknowledgments
This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (NRF-2021R1A2C2012855)

During sugery, various diseases can occur dut to abnormal blood vessel. Thrombosis and air embolism occurs frequently. Diagnosis of thrombosis and air embolism is CT or Doppler examination is typically used. These diagnosis are difficult to detect immediately during surgery and cost a lot of time and money. In this paper, a device capable of pinch grip propose to stable hold blood vessel without damage. The device has a capacitance sensor and crack-based strain sensor to measure a pulse wave and detect substance in the blood vessel. An in vivo experiment using the carotid artery of a pig was conducted to demonstrate the potential of use in bio medical device. As a result, the strain sensor has same pulse wave as commercial BP monitoring system. At the same time, it was cofimed through the C-arm that the capacitance sensor detects air embolism. It was possible to predict the occurrence of a stroke. In the future, the pinch gripper can be used as a biomedical device for a innovative blood vessel monitoring system in surgery. Our device can prevent dagerous complications such as hypotension, stroke, and heart attack in transplant and long time surgery.

High-performance biochemical sensors that can seamlessly adhere to the human body for long-term monitoring of physical and mental conditions are of rapidly growing interest during past years. Among the major configurations of biosensors, the transistor-type biosensor shows superior sensitivity owing to its internal amplification capability, and thus is regarded as the next generation of wearable and implantable biosensors. However, so far those biosensors still lack tissue-like mechanical properties that are highly compatible with the human body, such as softness and stretchability, due to the rigid and brittle nature of semiconducting materials. Here, we develop a new type of polymer semiconductor that can simultaneously achieve tissue-like softness and stretchability, together with high mobility. We further utilize the tissue-like semiconductors to fabricate transistors with desired mechanical and electrical performance for human integration, and demonstrate its capability as the advanced platform for biosensing applications.

The use of 3D printing scaffolds and interfaces has become popular due to its optimal customization and process time. Polylactic Acid (PLA) is a commonly used biocompatible and biodegradable filament material in 3D printing. However, PLA is naturally hydrophobic. Cell growth and cell adhesion on biocompatible polymers are affected by the surface energy properties and the interface. This study is focused on mitigating these problems by incorporating plasma micro-discharge surface modification to tailor the surface roughness characteristics of soft PLA and soft PLA composites toward the development of biocompatible substrates for enhanced cell adhesion and growth. There is limited research exploring how changes in plasma parameters affect the wettability and surface energy characteristics of materials. The current work involves the development of correlations between plasma properties and surface characteristics using machine learning models. Consequently, this work will also review the changes of 3D printed PLA in wettability, surface roughness, and surface morphology as the input voltage of the plasma micro-discharge treatment (confined micro plasma-based Townsend discharges) is varied.

The current work focuses on the development of a flexible non-invasive glucose monitoring device embedded in an active polymer matrix (e.g. PVDF-TrFE or PVDF-BaTiO3). The device consists of an embedded circuit with a pulsed laser and a continuous LED light in the near infrared range. This light source is incident on a glucose control solution to mimic a range of glucose concentrations in blood (in the range from 0 - 500 mg/dl). The glucose levels are detected by a perovskite-halide optoelectronic sensor (refraction) and are validated by a perovskite oxide transducer (photoacoustic) while using the active polymer matrix as a energy conversion media. Correlations between the output voltage and the glucose concentrations are developed using machine learning methods. The active properties of the polymer matrix is also characterized using electrical impedance spectroscopy.

Current standard clinical options for patients with detrusor underactivity (DUA) or underactive bladder -- the inability to release urine naturally -- include the use of medications, voiding techniques, and intermittent catheterization, for which the patient inserts a tube directly into the urethra to eliminate urine. Although those are life-saving techniques, there are still unfavorable side effects, including urinary tract infection (UTI), urethritis, irritation, and discomfort. Here, we report a wireless, fully implantable, and expandable electronic complex that enables elaborate management of the abnormal bladder function via seamless integrations with the urinary bladder. Such electronics can not only record multiple physiological parameters simultaneously, but also provide direct electrical stimulation based on a feedback control system. Uniform distribution of multiple stimulation electrodes via mesh-type geometry realizes low-impedance characteristics, which improves voiding/urination efficiency at the desired times. In vivo evaluations using live, free-moving animal models demonstrate system-level functionality.

Bioelectronic platforms that can combine electrophysiological and optogenetic/optical mapping techniques are becoming increasingly important tools as they can be used for the study of multifunctional electrical and optical manipulations with cell-type-specific resolution. However, conventional rigid or opaque electrodes have difficulties bridging these two technologies, as such platforms require a balance between transparency, flexibility, biocompatibility, and good electrical and electrochemical performance. For example, indium tin oxide (ITO) is the most widely used transparent conductive material in optoelectronics, but its inherent rigidity and brittleness limit its application in soft and curvilinear biointerfaces. Based on this, soft and transparent microelectrodes that can be integrated with soft tissue systems for biomedical and biological applications show great potential in multifunctional optical and electrical biointerfaces. Meanwhile, new materials and fabrication strategies are urgently needed to realize soft and transparent microelectrodes.

To meet the need, we provide three solutions. One is a hybrid structure microelectrode/interconnect using ITO and metal grids (ITO/metal grid), one is based on silver nanowires (Ag NWs)/SU-8 structure, and the other one is serpentine gold-coated Ag NWs (Au-Ag NWs)/SU-8 structure.

The ITO/metal grid structure exhibits high optical transparency (59-81%), excellent electrochemical performance (impedance: 5.4-18.4 Ω cm²) and sheet resistance (R_s: 5.6-14.1 Ω sq^-1). Furthermore, the structure shows a significant improvement in mechanical flexibility (5000 bending cycles at 5 mm radius) compared to pristine ITO (2 bends). Finally, the ITO/metal grid microelectrodes demonstrate high-fidelity recording of transgenic mouse hearts under co-localized optogenetic stimulation.

The Ag NWs/SU-8 structure exhibits excellent transparency > 90.0% at 550 nm and can stably withstand up to 100,000 bending cycles at 5 mm radius. Various concentrations of the Ag NWs enable tunable electrochemical (impedance: 3.4-15 Ω cm²) and electrical (R_s: 4.1-25 Ω sq^-1) performance. In addition, the solution-processing fabrication technique allows both large-area (10×10 cm²) and small-pattern (15 μm-wide grids of Ag NWs) fabrication. The flexible and transparent Ag NWs microelectrode arrays (MEAs) demonstrate the ability to record high-fidelity electrogram signals during optical mapping and co-localized optogenetic pacing.

Furthermore, in our third design, the chemical stability and electrochemical impedance of Ag NWs microelectrodes are significantly improved by coating a thin layer of Au (6 nm) with only slight changes in optical properties. The MEAs exhibit a high optical transparency >80% at 550 nm, a low normalized electrochemical impedance of 1.2-7.5 Ω cm², stable chemical and electromechanical performance after exposure to oxygen plasma for 5 minutes. The serpentine structure of Au-Ag NWs/SU-8 on stretchable polymer enables the device to withstand 600 cycles of stretching at 20% strain, superior to other transparent microelectrodes. The stretchable and transparent MEAs enable conformal integration with curvilinear heart surfaces for co-localized electrophysiological and optical mapping of cardiac function.

These proof-of-concept works indicate that the flexible ITO/metal grid, Ag NWs MEAs and stretchable Au-Ag NWs MEAs are promising candidates for the next-generation multifunctional biointerfaces for broad biological and biomedical research.

Cardiac arrhythmias as manifested by the disruption of normal electrical and contractile activities of the heart cause significant morbidity, mortality, and economic burden of health care every year. Cardiac minimally invasive and implantable electronic devices such as catheters, pacemakers, and defibrillators have been accepted as highly effective for clinical monitor and treatment of heart rhythm diseases via the electrical sensing and stimulation modalities. However, device mechanical rigidity leads to non-ideal interfaces with soft tissues causing various complications such as local pain, vascular injury, cardiac perforation, and infection. As a result, conformal devices melding to the curvilinear topology of the heart are desired.

Graphene possesses outstanding properties suitable for soft bioelectronics development such as sub-nanometer thickness, strength (130 GPa), stretchability (~15%), electrical conductivity, and biocompatibility. Meanwhile, the excellent optical transparency (~97% for visible light) of graphene further extends its application potential to optical studies which are nowadays critical tools (e.g., optical mapping and optogenetics) in the field of cardiac electrophysiology. Conformal and transparent biointerfaces combining the advantages of electro- and opto-techniques and achieving simultaneous multimodal sensing and/or actuating would therefore benefit both basic scientific research and clinics in studying and modulating the complex cardiac events.

In this work, we report for the first time on graphene biointerface developed via a cost- and time-effective, graphene electronic tattoos (GETs) based fabrication protocol for in vivo cardiac electrophysiology. Leveraging sub-micrometer thick tissue-conformable graphene arrays, we demonstrate sensing and stimulation of the open mammalian heart both in vitro and in vivo. Furthermore, we demonstrate graphene pacemaker treatment of a pharmacologically-induced arrhythmia, AV block. The arrays show effective electrochemical properties, namely interface impedance down to 40 Ohm×cm2 @ 1kHz, charge storage capacity up to 63.7 mC/cm², and charge injection capacity up to 704 µC/cm². Transparency of the graphene structures allows for simultaneous optical mapping of cardiac action potentials and optogenetic stimulation while performing electrical measurements and stimulation. Our report presents evidence of the significant potential of graphene biointerfaces for the future clinical device- and catheter-based cardiac arrhythmias therapies.