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. I will discuss a numerical model that trys to capture the essential nature of the photostimulation mechanism by numerically solving the Poisson-Nernst-Planck (drift-diffusion)  equation with proper boundary conditions taking into account theelectron transfer processes occuring at the interface. 
 

Currently, on-skin wearable or on-skin devices mainly rely on power sources such as non-rechargeable batteries, which are often toxic and harmful to the environment. Therefore, there is a strong need to develop new self-powered energy sources that possess human- and eco-friendly characteristics, including biocompatibility, degradability, and breathability. In this context, biofriendly and sustainable triboelectric nanogenerators (TENGs) emerge as excellent candidates for the next generation of green energy sources. TENG devices harness mechanical energy from various activities and environments, such as biomechanical motions and convert it into electrical energy. They are are cost-effective, easy to fabricate, and highly efficient, making them suitable for self-powered electronics and independent power source modules. Although there is a wide range of triboelectric materials available, many TENGs still rely on fluorinated polymers, which are known to be non-environmentally friendly and may cause discomfort and skin infections when used in on-skin wearables. To address these challenges, we demonstrate a cellulose-based tribopositive layer that enhances the TENG power output by introducing different surface functional groups through nano-coating or chemical/mechanical reduction to form nano-crystals or fibers. Additionally, we report on a new bio-polymer-based tribonegative layer, mimicking the chemical structure of conventional non-biodegradable materials. In the pursuit of a completely biodegradable TENG, we replace the substrate with a biopolymer film and employ carbon-based electrodes. These bio-TENGs offer flexibility, biocompatibility, and breathability, making them suitable for use as wearable devices that can harvest energy from biomechanical motions. Notably, these devices exhibit high performance with output voltage values surpassing 1 kV. This represents a twofold increase compared to conventional substrates and electrode materials employing polyimide as a negative triboelectric layer. Additionally, in the case of all-biopolymers-baed TENGs, the device output exceeds 500 V, which outperforms other reported bio-TENG devices in terms of voltage output generation. 

Bioelectronic devices show great promise in healthcare and environmental sensing due to their ability to provide longitudinal monitoring to maintain optimal status and evaluate physical and (bio)chemical changes. Electrochemical (bio)sensors are at the centre of this effort and offer a vast potential to revolutionize conventional diagnostics and environmental sensing methods that uses traditional laboratory tests-based evaluations, that are slow and mainly require in-person visits or frequent sampling if the long-term analysis is necessary.

In this presentation, I will give a brief overview of our recently developed electrochemical methodologies targeting of sensing and monitoring of various metabolites, hormones and microorganisms to acquire better knowledge on diagnosis and disease progression of diseases and environmental changes. This talk will summarize how to design sustainable sensors, and integrated electronics and how to use them in clinical and environmental settings with our unique access to patient materials and environmental samples, which creates an unprecedented opportunity to address fundamental questions in medical and environmental monitoring.   

The plant cell is protected by a complex polysaccharide cell wall composed primarily of cellulose, hemicellulose and pectin. This wall stabilizes a semi-permeable barrier, the plasma membrane (PM) that serves two main roles: (1) transport of essential molecules in and out of the cell, and (2) sensory transduction of environmental stimuli. Considerable progress has been made over the past two decades in understanding plant membrane proteins from a genetic perspective, but complementary tools for physiological, electrophysiological, and biochemical characterization necessary for studying protein functions are lacking. This work promotes a new cell-free, bioelectronic platform that captures the properties of green plant PMs and enables the measurement of plant transporter functions.

Monitoring the flux of metal ions across plant cell membranes through protein transporters embedded within them is a significant challenge today, but is fundamental for assigning function to unknown transporter genes; tackling transporter substrate specificities and modes of regulation; and eventually linking metabolic pathways to cellular compartments, plant growth and development, and bridging the genotype to phenotype gap. Today's technologies are inadequate for a number of reasons, including low throughput and lack of sensitivity, especially for transporters, which have fluxes several orders of magnitude lower than ion channels.

            In this presentation, I will share a new technology that combines planar green plant membranes with transparent, electrically conducting polymer electrodes for the dual-mode (optical or electrical) measurement of plant protein functions. Organic electrochemical transistors (OECT) based on the conductive polymer PEDOT:PSS offer biocompatibility, high-quality electrical signals, and optical monitoring due to the transparency of PEDOT:PSS thin films. We demonstrate here the application of the OECT to the measurement of copper transporter 1 (COPT1), which maintains plant copper homeostasis, but its function has not been wholly characterized. We formed a supported lipid bilayer (SLB) with membranes from transient GFP and GFP-COPT1-transfected Arabidopsis thaliana mesophyll protoplasts on the surface of the OECT. We measure copper flux through COPT1 in a specific and concentration-dependent manner. After data analysis, we obtain kinetic parameters of transport for COPT1 and report on the coupling of COPT1 with another protein, a copper reductase.

This new kind of plant-derived sensor device is amenable to scale up and we anticipate that it can be highly multiplexed for the collection of large data sets on plant transporter systems in a way that has not been possible before. With this capability, such large data sets can be fed into big data science approaches for enabling discoveries and breakthroughs in our understanding of how plants adapt to genetic perturbations, extreme weather conditions, pathogen pressures, and other critical aspects important for flourishing ecosystems.

The possibility to control living matter with exogenous stimuli can have tremendous impact on synthetic biology, medicine and materials science, among others. For instance achieving control over cells behaviour remains a challenge at the interface between living and non-living matter,^[1] and would enable the development of new bio-mimetic and bio-enabled materials able to perform tasks.^[2] Within this context, bacteria have arisen as “active and actively-controllable materials”, exhibiting neuro-like behaviour, extended bioelectric signalling^[3,4] and tunable assembly properties.^[5] In the last decade, it has been observed that the regulatory element of such an active behaviour is the electrical potential across the membrane, which governs bacteria electrophysiology, metabolisms and bioenergetics.^[6] Light can be a powerful tool in these regards, as one can control the membrane potential and, thus, cell function and behaviour remotely and with relatively high spatiotemporal precision.

Here, I will show that a membrane-targeted azobenzene can be used to photo-modulate precisely the membrane potential in cells of the Gram-positive bacterium Bacillus subtilis. We found that upon exposure to blue- green light, the isomerization reaction in the bacteria membrane induces hyperpolarisation of the potential (ΔV = 20 mV), within a bio-mimetic mechanism reproducing the initial fate of retinal. Apart from being promising results in the view to photocontrol bacterial motion and assembly behavior in consortia, this approach also highlights the role of previously uncharacterized ion channels in bacteria electrophysiology.^[7]

The interface between biological cells and non-biological materials has profound influences on cellular activities, chronic tissue responses, and ultimately the success of medical implants and bioelectronic devices. For instance, electroactive materials in contact with cells can have very different composition, surface topography and dimensionality. Dimensionality defines the possibility to have planar (2D), pseudo-3D (planar with nano-micropatterned surface) and 3D conductive materials (i.e., scaffolds) in bioelectronics devices. Their success for both in vivo and in vitro applications lie in the effective coupling/adhesion of cells/tissues with the devices’ surfaces. It is known how a large cleft between the cellular membrane and the electrode surface massively affects the quality of the recorded signals or ultimately the stimulation efficiency of a device. In fact, the shape and the composition of these newly designed electrodes recalls those features of neuronal cells to ultimately induce biomimetic recognition. Furthermore, conductive materials that exhibit dynamic properties and organic composition might better recapitulate the native environment of tissue. However, engineering such new platforms still require materials synthesis, integration into microfabricated platforms and stable cell interfacing. Here, we propose a new class of organic conductive materials which resemble the biomimetic architecture of neurite and dendritic spines, can undergo dynamic reshaping and can be functionalized with artificial membrane to enhance cell coupling and outgrowth. The cell response can be monitored and eventually is adapting to the substrate changes over time. These new electrodes can pave the way to adaptive microdevices of use in bioelectronic applications such as electrophysiology and sensing.

Traditional electronic devices are made of non-renewable and often toxic materials, which can lead to serious environmental contamination upon their disposal. A promising strategy for producing sustainable electronics is the direct writing of laser-induced graphene (LIG) electrically conductive patterns on biological and bio-based substrates. However, high ablation rates, the need to use controlled atmosphere, toxic fire retardants and multiple lasing steps limit the applicability of conventional LIG processes to produce sustainable electronic devices.

We have introduced iron-catalyzed laser-induced graphitization (IC-LIG) as an innovative technique enabling to engrave large-scale electrically conductive patterns on thermally sensitive substrates such as wood and wood-derived materials [1]. Our approach makes use of an aqueous bio-based coating, inspired by the historical iron-gall ink, which protects the wood surface from laser ablation and thermal damage while preserving its mechanical properties. Thanks to our approach, it is possible to engrave highly conductive (up to 2500 S m-1) patterns even on thinnest wood veneers (> 400 µm) and cellulose paper within a single lasing step in ambient atmosphere using a conventional CO2 laser setup.

Here, we explore further the possibilities offered by our IC-LIG approach by elucidating the catalytic effect of iron under different lasing conditions. We can control the iron-carbon composite structure at the nano- and microscale level, e.g., by tuning the concentration of iron in the ink. This, in turn, allows us to tune the iron phases in the resulting IC-LIG materials. By precisely controlling the laser parameters and setup we are able to fabricate bio-inspired surface patterns at the meso- and macroscale, controlling the structure as well as the electrical properties on a multiscale level, allowing us to fabricate sustainable electrodes for prospective uses as energy storage and electrochemical devices.

[1] C. H. Dreimol et al., Nat. Commun. 2022, 13, 3680 (12 pp.).]

In vitro studies have a significant impact on the progression of novel therapies, cancer and stem cell research, and the drug discovery process. However, the current standard for in vitro models primarily relies on two-dimensional (2D) cell cultures, which fail to fully replicate the intricate cellular architecture and response to microenvironmental cues. To address this limitation, three-dimensional (3D) cell cultures have emerged as more accurate representations of the in vivo cellular environment. Tissue-engineered scaffolds are particularly promising in this regard. However, existing technologies for assessing 3D cell cultures are predominantly limited to endpoint assays, offering only a snapshot of cellular behavior. In this study, our goal is to overcome this challenge by employing continuous electrical assessment of 3D cell cultures cultivated on electroactive composite scaffolds composed of poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PEDOT:PSS) and MXenes. The scaffold architectures are created using the freeze-drying technique, and we investigate the influence of different scaffold compositions on electrochemical performance, morphology, swelling, biocompatibility. By combining the electroactive properties of PEDOT:PSS with the distinctive characteristics of MXenes, we aim to develop scaffolds that not only support cell growth but also enable real-time, non-invasive monitoring of cellular behavior. Our study surpasses conventional optical-based assessments by introducing continuous electrical measurements as a means of tracking the dynamics of 3D cell cultures. The electroactive composite scaffolds facilitate the detection of electrical signals associated with crucial cellular activities like attachment, proliferation, and differentiation. To evaluate the performance of the electroactive composite scaffolds, we culture them with Human Dermal Fibroblasts and assess parameters such as cell attachment, viability, and growth. By integrating the benefits of 3D cell culture and continuous electrical assessment, our study aims to comprehensively characterize scaffold-cell interactions and their potential applications in tissue engineering and regenerative medicine.

Biofilm, the predominant lifestyle of all bacterial species, is a form of bacterial growth that enhances survival in wetted natural and artificial environments. The accompanying resilience, including decreased susceptibility to disinfectants and antibiotics, increases the need for prevention of biofilm formation, particularly on implanted medical devices and on food contact surfaces. This has prompted a revision of traditional methods and practices, re-evaluating them in relation to bacterial adherence and growth. The metabolism of bacteria growing in biofilms compared to those undergoing planktonic growth differs vastly. One important determinant is the redox state of the microenvironment in which bacteria exist. Sensing of the environmental redox state controls the switch between aerobic and anaerobic metabolism as well as bacterial virulence. We showed previously that electrically charged surfaces with high charge storage capacity, like PEDOT-based conducting polymers, can be used to modulate biofilm formation by altering the redox states of the surfaces [1, 2]. To gain better understanding of this finding, we performed an in-depth analysis of the process of biofilm formation on redox-challenged PEDOT:PSS surfaces using GFP-expressing Salmonella and the optotracer EbbaBiolight 680. The latter is a non-toxic fluorescent tracer that allowed us to monitor and quantify the production of extracellular matrix components (ECM) in real-time by semi-quantitative spectroscopy and microscopy [3]. Redox challenge performed in a fast-charging electrochemical setup promoted bacterial production of higher amounts of cells and ECM on oxidized PEDOT:PSS surfaces compared to their reduced counterpart. When electrodes were separated and charging was carried out with respect to a Pt electrode, the slower charging process resulted in increased cell numbers as well as increased amount of ECM on the reduced surfaces. When isogenic Salmonella mutants unable to produce the ECM components curli and/or cellulose were tested, none of them responded to the redox challenge. This demonstrates a novel use of surface redox potential to modulate ECM curli production and accordingly biofilm formation. Anti-biofouling surfaces directly affecting ECM production may find use in sensitising biofilm forming strains to the effect of antibiotics and antimicrobial compounds.

Leveraging the biocatalytic machinery of living organisms for in-vivo fabrication of functional bioelectronic interfaces enables seamless integration of devices in tissue and formation of biohybrid systems. Previously we have demonstrated that plants can polymerize conjugated oligomers in-vivo forming conductors within their structure. We showed that the polymerization is enzymatically catalyzed by endogenous peroxidases, and we developed a series of conjugated oligomers that can be enzymatically polymerized in physiological conditions. The conjugated polyelectrolytes integrate within the plant cell wall structure adding electronic functionality into the plant that is then explored for energy storage. Recently we demonstrated intact plants with electronic roots that continue to grow enabling plant-biohybrid systems that maintain fully their biological processes. The electronic roots are used to build supercapacitors and biohybrid circuits to power low power electrochemical devices. Furthermore, we have extended this concept into an animal model system. We demonstrated that Hydra, an invertebrate animal, can polymerize intracellularly conjugated oligomers in cells that expresses peroxidase activity. The conjugated polymer forms electronically conducting and electrochemically active domains in the µm range integrated within the hydra tissue. Our work paves the way for self-organized electronics in plant and animal tissue for modulating biological functions and in-vivo bio-fabrication of hybrid functional materials and devices.

Advancements in synthetic biology have pushed the frontier of materials science into the realm of engineering living materials. This presentation focuses on bioengineering strategies for coupling the electronic metabolisms of living cells with electrodes. Building on recent discoveries in the extracellular electron transfer mechanisms of S. oneidensis, our research focuses on the heterologous expression of key cytochromes in a facultative anaerobe (E. coli) [1-2]. We employ a combination of colorimetric and electrochemical techniques to characterize extracellular electron transfer in these engineered microbes. Interestingly, the bioengineered strains exhibit enhancements in both mediated and non-mediated extracellular electron transfer, with the extent varying with the specific combinations of expressed proteins. This platform was used to demonstrate enhanced electricity production for applications such as waste water treatment. Importantly, this work represents the first demonstration of the complete electron conduit from S. oneidensis fo bioeletcricity genertaion in a foreign cell. This demonstartion significantly broadens the range of exoelectrogenic microbes, unlocking new applications in microbial electronics across various fields such as biotechnology, environmental science, chemical synthesis, and energy conversion.

Inspired by biocatalysts in nature, artificial catalyst systems with behavior similar to or exceeding the activity of biocatalysts have been widely studied for different healthcare and environmental applications. In this work, a hybrid organic-inorganic approach is used to combine conducting polymer with inorganic materials for biocatalysis. Three different MoS2 nanosheets (MSNSs) were synthesized based on different methodologies. Methodology 1:  ammonium tetra thiomolybdate (NH4)2MoS4 and hydrazine hydrate (N2H4·4H2O) were sonicated and transferred to an autoclave for the hydrothermal process. Methodology 2: MoS2 nanosheets were synthesized through a hydrothermal method using sodium molybdate Na2-MoO4.2H2O and L-cysteine. Methodology 3: MoS2 nanosheets were prepared through exfoliating of bulk MoS2 in N, N-Dimethylformamide (DMF) by sonication process. The morphology, and structural information of MSNSs were analyzed by TEM, SEM, XRD and Raman spectroscopy. Initial results show excellent catalytic performance of the MSNs with different concentrations of hydrogen peroxide ranging from 1µM to 10M. The catalytic behavior of the MSNSs were then compared with the performance of hydrogen peroxidase within the same range of concentration values.

Eman Aljawi, Akbota Kurmangaliyeva, Muhammad Jamali, Bushara Fatma, Waqas Waheed, Dean Everett, Charalampos Pitsalidis, Anas Alazzam, and Anna Maria Pappa.

“Bioelectrocatalytic lab on chip device for infection biomarker monitoring”

Viruses can easily mutate, as evidenced with SARS-Cov2, hence prevention and early detection of infection seems to be the most reliable strategies to cope with pandemics. The current available diagnostics tools, such as real-time polymerize chain reaction (RT-PCR), are not compatible with early detection as they are time-consuming, expensive, don’t offer multiplexing, and don’t offer on-site detection. To tackle these issues, Lab-on-Chip (LoC) biosensors have emerged, overcoming the limitations of conventional methods. Electrochemical biosensors mainly consist of electrodes integrated into a platform to offer portable, on-site, cost-effective, specific, and fast detection. This project introduces a multiplexing LoC biosensor for the simultaneous detection of 4 infection biomarkers; glucose, lactate, hydrogen peroxide, and C-reactive protein (CRP) all in one device using biocatalytic enzymes for the three metabolies and an antigen for CRP as biorecognition elements. Optimization on the electrode materials in terms of bioelectrocatalytic performance leads to the optimum sensing capabilities while allowing for multiplexing on a chip. This work compbines LoC technologies based on microfluidics and the recent advancements in materials science, combining conducting polymers with 2D electronic materials for the electrode fabrication.