Symposium SB11—Photo/Electrical Phenomena at the Interface with Living Cells and Bacteria

The ability to establish successful and efficient extracellular electron transfer (EET) between bacteria and electrode surfaces is critical for the development of mediated microbial electrochemical technologies. Here, we describe a phenazine-based mediator system to facilitate electron transfer from the model bacterium Escherichia coli during glucose metabolism. Phenazine redox mediators were experimentally evaluated, demonstrating distinct mediated currents, dependent on mediator structure. Our results show that the choice of a mediator with the appropriate redox potential is not the single aspect to consider when rationally designing future mediator-based EET systems. We will discuss the use of density functional theory and multivariate linear regression for understanding the structural properties that control phenazine mediated EET.

In order to limit and revert the effects of climate change we must find a strategy to convert nearly inert CO₂ to value-added chemicals to close the carbon loop. CO₂ utilization has been industriously studied resulting in a variety of catalysts aiding to reduce CO₂. One auspicious approach integrating both solar energy generation and CO₂ reduction consists of pairing autotrophic whole-cell bacteria with semiconductor nanomaterials. Nanomaterials at length scales similar to those of bacteria absorb light and provide the microorganisms with the requisite energy to undertake CO₂ reduction. The microorganisms convert CO₂, N₂ and H₂O with unparalleled selectivity and low-substrate activation all while self-repairing and reproducing. We present on the evolution of photosynthetic semiconductor biohybrids and how we have built on the earliest demonstrations of the concept. Our enhanced ability to control the microenvironment in a silicon nanowire array, led to an increase in loading of acetogenic bacteria S. ovata which in turn improved CO₂ to acetate conversion. We were able to drive this reaction with solar power resulting in a 3.7% solar-to-chemical conversion. We have also undertaken a process to quicken the autotrophic metabolism of S. ovata via methanol adaptation yielding a 50% increase in CO₂ turnover rate. Furthermore, acetate can be upgraded by a secondary biocatalyst. Therefore, we have designed co-cultures to upgrade the acetate to value-added products. We demonstrate a co-culture between acetogenic Sporomusa ovata and diazotrophic Rhodopseudomonas palustris for tandem CO₂ and N₂ fixation to tunable value-added products including acetate, NH₃, nitrogenous biomass, and biopolyester. The dynamics between these two organisms and the products of the co-culture can be directed by electrochemical inputs. Lastly, we present on a strategy to generate sugars through abiotic CO₂ electrosynthesis. Copper nanoparticles and boron doped diamond electrochemically reduce CO₂ to glycolaldehyde and formaldehyde respectively which together form sugars including glucose. These sugars are used to culture Escherichia coli which can be engineered to produce chemicals and materials.

Electroactive bacteria, such as the model organism Shewanella oneidensis MR-1, can couple the oxidation of organic electron donors to the reduction of external solid surfaces, including minerals and electrodes, utilizing outer membrane multiheme cytochromes that carry charge from within the cell to external surfaces. In addition to transporting electrons across the cell envelope, the multiheme cytochromes can also facilitate long-distance (micrometer-scale) redox conduction along the outer membrane and across multiple cells bridging electrodes. While electroactive bacteria are commonly used in the construction of bioelectrochemical energy technologies, the development of more advanced living electronics has been challenged by the inability to control the size and shape of the conductive biofilms in relation to solid-state components. To overcome this challenge, we developed an optogenetic deposition strategy in which projected blue light patterns control the expression of a cell-surface adhesion protein in S. oneidensis and direct cell attachment to the illuminated areas of transparent conductive surfaces. Using this technique, we showed that the electrochemical activity of the engineered light-patterned biofilms remained comparable to that of the wild-type and scaled with controlled biofilm size. Furthermore, by depositing biofilms onto transparent interdigitated electrode arrays and performing electrochemical gating measurements, we confirmed that biofilm conduction current can also be tuned as a function of patterned biofilm size. Precise control of biofilm geometry allowed us to determine the intrinsic conductivity of living Shewanella biofilms for the first time. Thus, we have developed a facile technique to direct the deposition of living conductive biofilms; we anticipate that this approach will enable further innovations for both studying and harnessing bioelectronics, akin to the role that traditional photolithography played in the development of solid-state electronics.

Photosynthetic microorganisms are capable of interacting with light by specific proteins, called Reaction Centers (RCs), which are located into cell membranes and convert photons into charge separated states, in a process with efficiency close to 100%. Our work has demonstrated that the light harvesting capability of the RC extracted from Rhodobacter sphaeroides R26 bacterium can be ameliorated by covalently binding tailored molecular organic antennas [1-3]. RCs have been directly exploited in electronic and electrochemical devices [4], acting as photoactive systems in photoelectrochemical cells [5], photosensors [6] and photoactive transistors [7]. Interestingly, intact living photosynthetic bacteria cells can be also integrated in devices for bioelectronic applications [8]. However, the use of conductive artificial materials, like polymers or inorganic electrode surfaces, could lead to detrimental effects for the bacterial viability. Conversely, adhesive biomimetic polymers such as polydopamine (PDA) are promising biocompatible coating materials for application of Rhodobacter sphaeroides R26 bacterial cells in bioelectronics. Here, the approach is based on in situ polymerization of dopamine monomer in cell medium, with the PDA polymer self-assembling around cells, allowing them to thrive by light absorption and ensuring their interaction with conductive electrode surfaces. Polydopamine has been already successfully exploited to stabilize the interface between Rhodobacter sphaeroides RCs with electrodes, avoiding protein denaturation and facilitating the integration in electronic systems [9-10]. Preliminary electrochemical characterization also unveils that PDA layer around living bacterial cells adhered onto electrodes does not alter their viability and it does not hinder the diffusion of mediators and their capability to react at the electrodes.

[1] F. Milano, R.R. Tangorra, O. Hassan Omar, R. Ragni, A. Operamolla, A. Agostiano, G.M. Farinola, M. Trotta, Angew. Chem. Int. Ed., 51(44), 11019-11023 (2012).
[2] O. Hassan Omar, S. La Gatta, R.R. Tangorra, F. Milano, R. Ragni, A. Operamolla, R. Argazzi, C. Chiorboli, A. Agostiano, M. Trotta, G. M. Farinola, Bioconjugate Chem. 27, 1614-1623 (2016).
[3] S. la Gatta, F. Milano, G. M. Farinola, A. Agostiano, M. Di Donato, A. Lapini, P. Foggi, M. Trotta, R. Ragni, Biochimica et Biophysica Acta-Bioenergetics, 1860 (4), 350-359 (2019).
[4] A.J. McCormick, P. Bombelli, R. W. Bradley, R. Thorne, T. Wenzel , C. J. Howe Energy Environ. Sci., 8, 1092-1109, (2015).
[5] F. Milano, F. Ciriaco, M.Trotta, D. Chirizzi, V. De Leo, A. Agostiano, L.Valli, L. Giotta, M.R. Guascito, Electrochim. Acta, 293, 105-115 (2019).
[6] M. Chatzipetrou, F. Milano, L. Giotta, D. Chirizzi, M. Trotta, M. Massaouti, M.R. Guascito, I. Zergioti, Electrochemistry Communications, 64, 46-50 (2016).
[7] M. Di Lauro, S. la Gatta, C.A. Bortolotti, V. Beni, V. Parkula, S. Drakopoulou, M. Giordani, M. Berto, F. Milano, T. Cramer, M. Murgia, A. Agostiano, G.M. Farinola, M. Trotta, F. Biscarini, Adv. Electronic Mater. 6 (1), 1900888 (2020).
[8] F. Milano, A. Punzi, R. Ragni, M. Trotta, G. M. Farinola, Adv. Funct. Mater., 29 (21), 1805521, (2019).
[9] 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. 2000140, (2020).
[10] G. Buscemi, D. Vona, R. Ragni, R. Comparelli, M. Trotta, F. Milano, G. M. Farinola, Adv. Sustain. Syst., doi.org/10.1002/adsu.202000303 (2021).

Organic nanoparticles (NPs) are promising functional materials for the development of advanced photonic and biophotonic systems. We already demonstrated that blind laboratory rats treated with P3HT nanoparticles were able to recover vision highlighting the potential biomedical applications in the context of neuronal stimulation and neuroprostheses [1]. Here, we investigated thiophene-based core@shell NPs, made of poly(3-hexylthiophene) (P3HT) in the core and oxygenated P3HT (PTDO) in the shell [2], proving their role as highly effective photon nanotransducers. This structure supports long-lived charge separation, originating from the type II alignment at the core/shell interface. Here we provide a general photophysical picture in accordance with the morphology and the oxidation degree of the nanoparticles. Atomic force microscopy and Kelvin probe force microscopy allowed to characterize the nano-organization within the NPs core@shell configuration and their surface photovoltage characteristics. Steady-state and time-resolved spectroscopic measurements suggest that energy transfer is taking place form the core to the shell, followed by charge separation at the surface.
Living cells photostimulation can be achieved exploiting different physical mechanisms such as chemical reaction, current injection, capacitive coupling and thermal effect. Each mechanism can be ascribed to a specific material or a particular geometry [1]. The application of these core@shell systems may advance our understanding of the coupling mechanism at the biotic/abiotic interface.

References
[1] Maya-Vetencourt, J.F., Manfredi, G., Mete, M. et al. Subretinally injected semiconducting polymer nanoparticles rescue vision in a rat model of retinal dystrophy. Nat. Nanotechnol. 15, 698–708 (2020)

[2] Di Maria F, Zanelli A, Liscio A, Kovtun A, Salatelli E, Mazzaro R, Morandi V, Bergamini G, Shaffer A, Rozen S, ACS Nano 11, 2, 1991–1999 (2017)

The possibility to control selectively the electrical activity of living cells with low invasiveness has opened up new therapeutic paths in neurodegenerative medicine. Optical technologies are particularly suited for this purpose, as light enables precise and localised perturbation of cell activity in a remote and spatiotemporal precise manner. In particular, cell membrane potential modulation occurs via the photoinduced modification of the membrane electrical properties, either through direct photostimulation or by using selected transducers that are able to convert light into an electrical, mechanical, chemical, or thermal stimulus. In these regards, organic semiconductors have emerged as powerful photoactuators for the development of functional interfaces with living cells and organisms, thanks to their relatively high absorption coefficient and to their bio-mimetic nature. Although many efforts have been dedicated to investigate the biophysics underpinning photostimulation with organic semiconductors, the exact stimulation mechanism is still matter of debate.
To shed light into these aspects, we employed an all-optical spectroscopic approach, based on the use of well-established fluorescence probes, to monitor the membrane polarity, viscosity and order directly in living cells under thermal excitation transduced by a photoexcited polymer film. Briefly, our findings demonstrate that the opto-capacitive process induced by temperature is not simply due to a geometrical effect, as it is commonly believed. Indeed, our results suggest that a thermal induced phase transition promotes water penetration into the disordered phase of the plasma membrane. Thus, we speculate that the resulting change in dielectric response of the plasma membrane is the ruling phenomenon explaining the temperature induced enhancement in capacitance. Furthermore, we find experimental evidence that optostimulation mediated by a photovoltaic organic film brings about an additional phenomenon reinforcing the perturbation effect. We tentatively assigned this to the surface charging at the bio interface, suggesting that optostimulation mediated by organic semiconductors is not simply due to thermal effects, but it is also related to the capability of the photogenerated charges to polarize the plasma membrane.

The development of a fully organic photovoltaic prosthesis of poly(3-hexyltiophene) (P3HT) for subretinal implants was proposed to treat degenerative blindness (Maya-Vetencourt et al.,2017 and 2020). Although the efficiency of organic polymer-based retinal devices in vivo has been proven, the interpretation of the working mechanisms that grant photostimulation at the polymer/neuron interface is still a matter of debate. In order to contribute solving this issue, we have first focused on the characterization of the interface between P3HT films and watery-electrolytes by the combined use of electrochemistry and mathematical modeling.
In particular, the electric behavior of P3HT is modeled through a system of time dependent Drift-Diffusion non linear partial differential equations. The model takes into account the working principles of the organic semiconductor in contact with the electrolyte solution, with a detailed description of its interaction with the dissolved oxygen. This effect experimentally proved to be crucial in the photoelectrical switch of P3HT.
In order to compare and validate the results of our mathematical model, we have performed several electrochemical measurements of photovoltage on P3HT film deposited onto an ITO substrate samples, in different oxygenating conditions and with active layers of various thicknesses. Mathematical simulations well reproduce the experimental results upon illumination of the device in the various conditions.
Besides validation, the good agreement between measurements and simulations can be used to highlight the main effects, secondary to the illumination of the P3HT device, necessary to reproduce the experimental behaviour. Upon illumination, the essential unipolar transport in the photoexcited film leads in few µs to a drift-diffusion equilibrium associated to a space charge separation, responsible for the initial negative photovoltage. Electron transfer reactions towards oxygen at the polymer/electrolyte interface extract negative charge from the polymer and are responsible for the positive signal that we observe after several ms. The quantitative description of the phenomena also permits the estimation of the charge accumulated at the interface, possibly responsible for an electrostatic interaction with a biological system.
In spite of the simple model studied, all these considerations shed light on the possible coupling mechanisms between the polymeric device and the living cell, supporting the hypothesis of pseudo-capacitive coupling arising from an electrostatic interaction between the polymeric film and the watery environment. In addition to this, once the fundamental mechanisms of P3HT have been quantitatively reproduced and formalized, it is possible to start developing models to reproduce the effects of P3HT nanoparticles in a watery-electrolyte, a system whose electric properties are hard to characterize experimentally but which appears to be the new frontier of retinal devices.

Nanoscale and microscale ZnO particles are utilized in a wide variety of applications due to their optoelectronic properties and high biocompatibility. One application is the use of ZnO as an antibacterial agent. Antibacterial action of ZnO is well documented, exhibiting growth inhibition for gram-positive and gram-negative bacteria as well as antibiotic resistant bacterial strains. In spite of considerable amount of research in this field , there is no clear consensus on the fundamental mechanisms behind this phenomenon. Suggested mechanisms include: cell internalization of the ZnO particles, release of Zn ions, generation of different reactive oxygen species, surface-surface interactions, etc. In our work, we address, in conjunction with the antibacterial assays, surface and near-surface optoelectronic properties of ZnO microcrystals participating in those assays. In particular, we look at the influence of crystalline morphology and crystallographic polarity on the interaction between bacterial cells and ZnO surfaces. To produce our antibacterial specimens we utilized a bottom-up hydrothermal growth method to synthesize ZnO microcrystals of tunable morphology and relative abundance of polar and non-polar surfaces. Scanning electron microscopy, energy-dispersive X-ray spectroscopy and surface photovoltage experiments were used to characterize the quality and morphology of the ZnO microcrystals. To establish the antibacterial activity of our particles, we ran minimum inhibitory concentration assays of staphylococcus aureus. By utilizing ZnO crystals larger in size than the s. aureus bacteria cells, we confirmed that internalization of the ZnO particles is not necessary to inhibit the growth of the bacteria. The larger microcrystals exhibited inhibition of bacterial growth comparable to that of nanoscale ZnO particles. To determine the influence of structural and surface properties of ZnO on the assay outcomes, we performed a series of optoelectronic experiments using photoluminescence spectroscopy as well as spectroscopic and transient surface photovoltage. These investigations were performed on ZnO crystals prior to and following exposure to s. aureus. Our results revealed significant spectral changes due to interactions with bacteria and growth media. We demonstrated that growth media have a significant impact onto the nature and efficacy of antibacterial activity of ZnO.

The intrinsic optical properties of proteins (i.e., absorbance at 280 nm and fluorescence around 340 nm) are known to originate from the electronic transitions of aromatic amino acids, such as tryptophan. As aromatic amino acids are present in nearly all proteins, intrinsic absorbance and fluorescence have limited use in protein characterization. Recently however, a new weak intrinsic absorbance signal has been uncovered in the visible region, even in proteins which lack aromatic amino acids. This previously overlooked visible absorbance signal has been theorized to arise from charge transfer states which form between charged amino acids within the protein. The unique interactions between charged amino acids likely affect this new intrinsic absorbance signal, and could be used to identify specific protein structures or sequences. Despite experimental observation of this new optical state, the exact photophysical mechanism has not yet been elucidated spectroscopically. Here, we characterize this newly uncovered optical state using advanced spectroscopy techniques in three highly charged proteins. The absorbance spectra of each protein was carefully measured using an integrating sphere to minimize contributions from scattering. We then used transient absorption spectroscopy to fully characterize the energy transfer within each protein, uncovering states with charge transfer character, consistent with theoretical simulations. By fully understanding this intriguing new charge transfer state, protein absorbance could eventually find use as an intrinsic probe for in vivo optical microscopy, or as a new method for the identification of specific protein conformations.

Cell therapeutics is a rapidly growing medicinal innovation being applied to cancer immunotherapy and degenerative diseases among others. Conventional pharmaceutical standards dictate a regulatory need to understand each and every cell placed within a patient. Thus, cellular therapeutic elements need to be monitored and assessed through various cell production process step to keep consistent and safe clinical outcome. However, this necessarily requires a new generation of non-destructive and label-free techniques that can characterize a single cell and their product at high-throughput, but otherwise leave it unadulterated before administration to the patient. Such a tool would bring unprecedented spatiotemporal resolution to intracellular and pericellular biochemical interactions at the scale of the fundamental building block of life with significant high-throughput. The scientific challenges include the need to measure at a rate and magnitude commensurate with the size of cell population and this goal has remain elusive over the past decades.
In this research, we utilize a power of nanosensor array and develop a new class of cell population monitoring platform that addresses those limitations. An array of near infrared (nIR) fluorescent single walled carbon nanotube (SWNT) nanosensors are integrated along a microfluidic channel through which a population of flowing cells will be guided, using specific integration technique of particulate form of fluorescent nanosensors. We show that one can utilize the flowing cell itself as highly informative Gaussian lenses projecting nIR emission profiles and extract rich information on a per cell basis in real-time and a non-destructive manner. This unique biophotonic waveguide allows users to quantify the cross-correlation of the biomolecular information with physical properties such as cell size, eccentricity and refractive index (RI). We show that it operates as a label-free chemical cytometer for the measurement of cellular heterogeneity with unprecedented precision for various type of cells and their target analytes. Our platform can be exploited to new class of chemical cytometry tools for cell-based therapeutic applications, where there are urgent needs for high-throughput characterization of biochemical properties cellular targets with therapeutic potential for new treatments.

The overarching goal of our research is to manipulate the light-bio-matter interaction to obtain control over the functioning of living systems. Light can control cell activity, with high space and time resolution and a virtually infinite number of configurations, free from wiring constrains. We develop and study non-genetic cell opto-stimulation techniques based on artificial light actuators that establish functional abiotic-biotic interfaces able to transduce a light signal into a biological stimulus. This talk reports on the state of our research regarding organic bio interfaces for inducing light sensitivity in cells, both in vitro and in vivo. Light actuators comes in different shapes: planar patches, nanoparticle, intra-membrane probes. Their coupling mechanism is still far from being understood and attempts to shed light will be introduced. The research aims at a new technological platform for application in life enhancing technologies or new cyborg technologies. One of the most appealing application of this emerging technology is rescue vision in blind people.

Human induced pluripotent stem cell-derived brain organoids have shown great potential for studies of human brain development and neurological disorders. However, quantifying the evolution and development of electrical functions in brain organoids is currently limited by measurement techniques that cannot provide long-term stable three-dimensional (3D) bioelectrical interfaces with brain organoids during development. In this lecture, I will present the cyborg brain organoid platform, in which 2D progenitor or stem cell sheets can fold “tissue-like” stretchable mesh nanoelectronics through organogenesis, distributing stretchable electrode arrays across 3D organoids. We previously demonstrated this technique in cardiac organoids, and I now expand it to two types of brain organoids, early-stage brain organoids, where the mesh nanoelectronics is integrated to the tissue culture before differentiation and organogenesis, and more mature brain organoids, where the mesh nanoelectronics is integrated after four months of differentiation. I will demonstrate that the tissue-wide integrated stretchable electrode arrays do not interrupt neuronal differentiation, adapt to the volume and morphological changes during organogenesis, and provide long-term stable electrical contacts with neurons within brain organoids during development. Then, I will show that the seamless and non-invasive coupling of electrodes to neurons enables a 6-month continuous recording of the same brain organoids and captures the emergence of single-cell action potentials from early-stage brain organoid development. The increase of action potential amplitude, synchrony, and firing rate as well as narrowing of action potential duration suggest changes in neural network connectivity and cellular ion channel expression level. Finally, I will discuss the integration of cyborg brain organoid platform with organoid-wide connectomics and spatial transcriptomics mapping to further test this hypothesis. Furthermore, cyborg brain organoid technology can potentially become a useful tool for quantifying the functional development of brain organoids and standardizing the culture conditions across different types of protocols by tracing organoid-wide tissue and single-cell electrical activity during the entire organoid development. It will also be useful for studies of developmental neuroscience and drug screening.

Nowadays stimulation and control over cellular activity and physiology is an emerging and hot topic. In particular, contactless and wireless methods are extremely appealing due to their ability to leave unaltered the cell and tissue condition. Following this idea, light represents a clean and spatiotemporal precise tool to achieve effective bio-stimulation. Light-transducers, such as conjugated molecules and macromolecules, have proven their efficacy at the interface with living cells and tissue. This interaction is possible thanks to their high optical absorption/emission, biocompatibility and versatility in chemical synthesis.
In this work, we employ a lipid membrane-targeted photochromic molecule to photomodulate signalling in cardiac cells. We characterized the single cell-molecule interaction, to evaluate its ability to photocontrol electrophysiological and functional behaviours. The ultimate goal of this project is the realization of a bio-hybrid photoresponsive smart tissue for future applications in robotics and pharmacology, as well as for tissue regeneration.

All cells, including bacteria, maintain an ion gradient across their membrane. This ion gradient results in a resting membrane potential that is essential to cell health. The use of light or electricity to control the resting membrane potential, compared to solution-based reagents, is unique in providing spatial control. We have designed a fluorescence microscopy-based platform to monitor and control membrane potential, while also imaging cell elongation and division. Our recent work has focused on the relationship between resting membrane potential and cellular elongation. Resting membrane potential is controlled by exposing B. subtilis cells to blue light, thereby limiting the effect to a subset of cells. Neighboring cells, not exposed to blue light, are used as a control. The elongation rate is measured for each cell, thereby incorporating the heterogeneity of cellular populations into each measurement. Our experiments distinguish two populations of cells with correlated membrane potential and elongation rates. The ability to modulate the resting membrane potential with light, and thereby control the growth of a subset of bacteria within a larger population, provides a method to better understand the fundamentals of bioelectricity and ultimately modulate cellular growth in a spatially-controlled manner.

In vitro models of biological systems are essential for our understanding of biological systems. Many difficulties exist in interfacing complex, biologically relevant models with technology adapted for monitoring function. Polymeric electroactive materials and devices can bridge the gap between hard inflexible materials used for physical transducers and soft, compliant biological components. In this presentation, I will discuss our recent progress in adapting conducting polymer devices, both OECTs (organic electrochemical transistors) and electrodes, to integrate with both mammalian and bacterial cell membranes supported lipid bilayers, preserving native membrane function. The conducting polymer devices preserve native membrane function, including transmembrane protein function, but also provide a means to readout changes to membrane properties such as permeability or binding or fusion with the membrane. Since the cell membrane is the gateway to the cell, this platform allows monitoring of pathogens such as viruses that may enter the cell, or drugs or toxins that may interact with the membrane compromising normal function (e.g. toxins binding to ion channels in the membrane or antibiotics that disrupt bacterial membranes). In this presentation I will describe the development of this platform and showcase how it may be used as a rapid and highly quantitative method for monitoring events occurring at the membrane in a robust and scalable manner without recourse to live cells.

Charting single-cell gene expression and electrophysiology in intact three-dimensional (3D) tissues across time and space is crucial to fields ranging from developmental biology to cardiology and neuroscience. Such multimodal methods require stable and continuous recording of individual cell electrical activity with high spatiotemporal resolution across 3D tissue, multiplexed profiling of a large number of genes in electrically recorded cells, and cross-modal computational data integration and analysis. In this talk, I will discuss 1) “tissue-like” electronics that possess tissue-like properties for long-term stable single-cell electrophysiology in tissues, organs, and behaving animals; 2) “cyborg organoids”, where stretchable sensor arrays are embedded in 2D sheets of stem/progenitor cells and reconfigured through 2D-to-3D organogenesis, enabling 3D electrophysiology over the time course of organoid development; and 3) “in situ electro-sequencing” that integrates “tissue-like” electronics, in situ sequencing, and machine learning to chart cell electrophysiology and gene expression at single-cell resolution across time and space.

Filamentous multicellular cable bacteria facilitate long distance electron transport (LDET), up to centimeter scales, in vast marine and freshwater sediments, indicating that LDET is an essential process in global geochemical cycling.
Through this process, cable bacteria couple sulfide oxidation by cells that dwell in the deeper sulfidic zone to oxygen reduction by cells near the surface sediment. Recent electronic measurements of cable bacteria enriched from marine sediments demonstrated that the electron transport pathway consists of a network of periplasmic fibers that run along the unique cell envelope of entire cables. Here we focus on demonstrating and understanding the electron transport characteristics through cable bacteria from freshwater sediments.
Long cable bacteria filaments were recovered from incubations of freshwater lake sediments (Ballona pond and Mungi Lake) in Southern California. The Southern California Freshwater cable bacteria share >97% sequence similarity to the 16S rRNA gene sequences from the freshwater cable bacterial genus Candidatus Electronema within the Desulfobulbaceae family. Current-voltage measurements of cable bacteria bridging 3-mm gaps between interdigitated electrodes reveal nano- to microampere currents through intact single filaments. A combination of atomic and electrostatic force microscopy measurements revealed the characteristics of the underlying periplasmic fiber network, as previously described in marine cable bacteria. In addition, conductive atomic Force microscopy was used to directly assess the conductivity of locations on individual cells. Using four-probe electronic measurements, the conductivity of single fiber in freshwater cable bacteria filaments is found to be ca. 0.1 S/cm. Our study expands the knowledgebase of centimeter scale LDET in cable bacteria to the freshwater niche and shed light on the mechanisms underlying their remarkable conductivity.

Exosomes are nanosized, lipid bilayer-delimited particles that act as mediators in cell-cell communication and have the ability to transfer RNAs and proteins between donor and recipient cells to elicit a variety of cellular responses. Tumor cell-derived exosomes (TDEs) have recently been reported to play an active role in tumorigenesis and metastasis owing to their ability to transmit oncogenes. One hypothesis is that TDEs contribute to metastasis by inducing the epithelial-to-mesenchymal transition (EMT), characterized by the loss of barrier function in barrier tissue-forming cells.

This study aims to elucidate the role and mechanisms of TDE uptake by investigating the process by which TDEs “infect” normal cells inducing EMT prior to metastasis. Current strategies for investigating the spatial and temporal aspects of exosome uptake, often optical, lack the ability to obtain quantitative data in real time. We have shown that organic electronic devices based on the conducting polymer poly(3,4-ethylenedioxythiophene): poly(styrenesulfonate) (PEDOT:PSS) can be interfaced with biological systems of varying complexity allowing for quantitative real-time monitoring of biological interactions. The optical transparency of our PEDOT:PSS-based devices provides the unique advantage of dual transduction increasing the credibility of our platform and enriching the biological information obtained. Moreover, their compatibility with microfabrication methods allows for high throughput studies.

We have shown that TDEs derived from the triple-negative breast cancer cell line MDA-MB-231 can induce an EMT-like process in MCF-10A non-tumorogenic breast epithelial cells as demonstrated by changes to cell morphology, loss of apico-basal polarity, reorganization of filamentus actin, and gain of mesenchymal proteins, such as vimentin and N-cadherin. An integral feature of EMT is the dissolution of tight junctions and the consequential loss of lateral cell-cell adhesion leading to a phenotype with increased motility and invasiveness. In ongoing studies, this decrease in barrier integrity of the cell monolayer is monitored electrically using organic electrochemical transistors, gaining of truly quantitative insight into TDE-induced EMT with higher temporal resolution than conventional, orthogonal methods. This data will be further substantiated by monitoring the expression of immunofluorescently labelled barrier-forming tight junction proteins. These combinatorial optical and electrical measurements of TDE interactions with cell monolayers provide invaluable information for developing strategies that may inhibit TDE interactions by e.g. blocking specific surface markers, thereby preventing TDE-induced EMT with implications for preventing cancer metastasis.

The viscosity of cell membranes is a crucial parameter that affects the diffusion of small molecules both across and within the lipidic membrane and that is related to several diseases, such as atherosclerosis, Alzheimer's, diabetes, and cell malignancy. In general, the precise determination of viscosity at the molecular scale represents itself an important issue in cellular biophysics. Despite the great interest of viscosity measurements on the nanoscale, it is still challenging to retrieve reliable values, as the traditional mechanical methods used for probing viscosity are not suitable for living cells.
We investigated the photophysics of a recently synthesized amphiphilic membrane-targeted azobenzene (ZIAPIN2) and we validate its use as viscosity probe for cell membranes. Indeed, we proved that the possibility of ZIAPIN2 of undergoing trans-cis photoisomerization depends on the viscosity of the environment. This led to the development of a molecular viscometer based on time-resolved fluorescence spectroscopy, that we used to assess the viscosity of Escherichia coli bacteria membranes. Lifetime measurements of ZIAPIN2 in E. coli bacteria suspensions correctly indicate that membrane viscosity decreases as the samples were heated up. Our results estimate a membrane viscosity value in living E. coli cells going from 10 to 5 cP, increasing the temperature from 22 °C up to 40 °C.

The resting membrane potential regulates bacterial division, elongation, and electrical signaling. Use of an external electric field is an effective method to regulate these properties with the advantage of providing spatial control, altering only a subset of cells. The challenge is to develop devices for precise control of the applied electric field with simultaneous monitoring of membrane potential and bacterial growth. Our initial research developed a device using two gold wires as electrodes to apply an electric field while imaging, with either brightfield or fluorescence microscopy, bacteria, including a control population in the absence of an electric field. Although useful for initial studies of membrane potential-regulated bacterial elongation and division, this device was low throughput. A second-generation device has been designed and fabricated by photolithography. The gap distance between the two electrodes, which determines the electric field, can be controlled from 10 μm to 1 mm, largely expanding the range of electric fields that can be achieved. Three pairs of electrodes with different gap distances can be integrated into the same device, increasing experimental throughput. Multiple electrode materials, including gold and platinum, can be used for these devices. In addition, the shape of the electrodes can be customized to achieve the desired electric field distribution. With this device, we can control the frequencies, amplitudes, and waveforms of applied electric fields while simultaneously imaging bacterial growth and membrane potential. We use this single cell-level imaging to understand and control heterogeneity of bacterial cells. Within any population of cells, individual cells will have different membrane potentials, which change in time, even in the absence of an applied electric field. This device allows us to track the membrane potential of individual cells over hours to address fundamental questions of bacterial electrophysiology.

The growing interest in the development of minimally-invasive therapeutic strategies has opened the doors to the introduction of one (or several) therapeutic needle(s) inside the tumor target that aim at inducing a controlled change of tissue conditions and environment to achieve tumor necrosis and apoptosis. Amongst all the treatment methods, ablative photothermal therapies are emerging as potential alternatives for the management and treatment of the tumor of which the laser-induced heated region are enabling the use of even finer ‘needles,’ where the spatial resolution can exceed the diffraction limit. Thus, studying the nanoscale heat transfer mechanisms within the target tissues and across their respective interfaces to gain a fundamental understanding of the dominant mechanisms within this class of therapies are critical for developing the next-generation of thermal ablation treatments.

In this study, we experimentally investigate these fundamental heat transfer mechanisms that dictate photothermal therapy efficiencies in nanoparticle-mediated systems. In particular, we employ ultrafast time-domain thermo-transmission (TDTT)¹ measurements to monitor the heat flux from heated nanoparticles directly into surrounding biological cells. Further, by implementing a tunable-wavelength mid-infrared probe to these measurements, these measurements can directly resonate with different vibrational modes of interest, thus providing insight to which modes dictate the interfacial heat transfer from nanoparticles into surrounding biological tissues. In this study the biological parameters on the temperature distribution, the heat penetration depth, and degree of irreversible damage as a result of the heat source parameters are investigated. These experimental measurements provide direct insight into the vibrational modes that dictate interfacial heat transfer during photothermal therapies and the means of which material parameters can be tuned to enhance these heat transfer pathways.

1. Jung, H., Szwejkowski, C. J., Pena-Francesch, A., Tomko, J. A., Allen, B., Ozdemir, S. K., Hopkins, P., and Demirel, M. C., ‘Ultrafast laser-probing spectroscopy for studying molecular structure of protein aggregates,’ Analyst 142, 1434-1441 (2017)

A multitude of microbes have adapted to gain energy by transferring electrons to external abiotic surfaces ranging from minerals to solid-state electrodes. In the model metal-reducing bacterium Shewanella oneidensis MR-1, this extracellular electron transfer (EET) is achieved through a network of multiheme cytochromes that span the cell envelope and incorporate along microbial nanowires. Recent studies of biomolecules, including cell surface electron conduits, have shown that electron transport in these proteins is spin selective through the chirality induced spin selectivity (CISS) effect. However, the physiological impact of CISS on electron transfer by living cells remains unclear. To further investigate CISS, we perform Hall effect measurements to screen for spin filtering in the S. oneidensis periplasmic Small Tetraheme Cytochrome (STC), which bridges the periplasmic gap between inner-membrane components and the cell surface electron conduits. STC has one of the highest heme-to-volume ratios of any known proteins, resulting in closely packed hemes and fast electron transport rates. By performing spin polarization measurements of an STC variant engineered to better interact with electrodes we show that, similar to previous reports of the cell surface cytochromes MtrF and OmcA, electron transport through STC is also spin selective. Our study provides an improved understanding of the electron transport physics in biomolecules and sheds light on an intriguing phenomenon whereby biology may exploit both electron spin and charge for interacting with abiotic surfaces.

Efficient, multi-enzyme cascade biocatalysis is currently an unmet challenge, with a high potential in industrial biofuel cell applications. To address this issue, we developed a single-step exfoliation combined with enzyme binding by simply stirring the enzymes and commercially available, inexpensive, multi-walled carbon nanotubes (CNTs). This resulted in highly active biocatalysts that showed very high activities, greater than those of the free enzymes, under otherwise identical conditions of pH, temperature, and ionic strength. This major breakthrough is reported here, where bovine serum albumin (BSA) is used as a co-exfoliant to assist in the formation of the biocatalytic dispersions of CNT/BSA/glucose oxidase (GoX)/horseradish peroxidase (HRP) complexes. The BSA not only promotes exfoliation of CNTs but also passivates the high energy sites on the CNTs and protects the delicate enzymes from denaturation on the hydrophobic surface. The CNT/BSA/HRP complex showed ~2 times higher activity than that of the free HRP, and the CNT/BSA/GOx/HRP showed 6-times greater activity than that of the corresponding mixture of the free enzymes, under otherwise identical conditions. We hypothesize that the CNT surface couples the active sites of these two redox enzymes, promoting the overall activity of the enzyme cascade events. Hence, the biological CNT enzyme complex forms a complex interface, achieving efficient and robust binding while preserving the biological activities of these delicate enzymes. Further, the cascade enzymes are being developed for the construction of industrial-scale biofuel cells. Thus, an improved, simple method is developed to produce highly active cathodes that can be readily adapted even under resource-limited conditions.

Keywords: Carbon nanotubes, proteins, biocatalysts, biofuel cells

A highly localized and effective, minimally invasive, physiologically stable and wireless electro-stimulation capability would present an important instrument in the toolbox of bioelectronic and electrophysiological methods. It is a milestone in the development of bioelectronic therapies, neural prostheses such as artificial retinas, artificial limbs and auditory prostheses, and brain-machine interfaces. Photo-capacitive organic electronics-based bioelectronic devices – organic electrolytic photocapacitors (OEPC), have demonstrated effective, chronic and localized in-vivo wireless peripheral nerve electric stimulation¹, as well as in-vitro wireless single cell² and retina electrostimulation³. Planar OEPCs fabricated on micrometer-thin flexible substrates have enabled conformal adhesion of the devices to the target tissue. Furthermore, a choice of organic pigment-based semiconductor PN bilayer has enabled effective and stable long-term operation of the devices in-vivo. The bioelectronic interface of an OEPC consisting of a circular-shaped PN bilayer as the active electrode deposited on top of an extensive, transparent conductor comprising the back electrode has been shown to stimulate peripheral nerves effectively. However, the simple circular shape of the top electrode in conjunction with the planar back electrode disperses the stimulating electric currents radially, which may not present the optimal use of the photogenerated charge in applications where high charge density and spatial localization are essential. Similar reasoning can be applied to various trivial electrode shapes, which are often used elsewhere.
We will show how spatial planar and 3D structuring of the OEPC’s PN and back electrode layers affects their optoelectronic, bioelectronic and mechanical properties, simultaneously shaping their functionality. We will demonstrate how a complete 3D numerical model comprising the excitable tissue in contact with the photo-capacitive device can be used to tailor the effects of an optically driven device at the interface with living cells. We have manufactured 3D and planar OEPC devices with spatially optimized interfaces, which showed multifold improvements in performance compared to the trivial geometries previously used.

[1] A chronic photocapacitor implant for noninvasive neurostimulation with deep red light, M. Silvera-Ejneby, M. Jakesova, J. J. Ferrero, L. Migliaccio, Z. Zhao, M. Berggren, D. Khodagholy, V. Derek, J. Gelinas and E. D. Glowacki, bioRxiv (DOI: 10.1101/2020.07.01.182113), accepted for publication (Nature Biomedical Engineering, June 2021)
[2] Optoelectronic control of single cells using organic photocapacitors, M. Jakesova, M. Silvera-Ejneby, V. Derek, T. Schmidt, M. Gryszel, J. Brask, R. Schindl, D. T. Simon, M. Berggren, F. Elinder, E. D. Glowacki, Science advances, 5 (2019) eaav5265
[3] Direct Electrical Neurostimulation with Organic Pigment Photocapacitors, D. Rand, M. Jakesova, G. Lubin, I. Vebraite, M. David-Pur, V. Derek, T. Cramer, N. S. Sariciftci, Y. Hanein, E. D. Glowacki, Adv. Mater. 30 (2018), 1707292

Plasma membranes represent a biophysical barrier separating the cytoplasm from the extra cellular matrix across living organisms. Their heterogeneous structure consists of a lipid bilayer embedded with proteins and other vital components, which largely determine the interaction of cells with their environment. Recently our group, in collaboration with the Daniel and the Salleo groups, have demonstrated that authentic plasma membranes in the form of supported lipid bilayers (SLBs) can be formed on top of poly(3,4-ethylenedioxythiophene) polystyrenesulfonate (PEDOT:PSS) based transducers. The bilayers were produced by a vesicle fusion process of native extra-cellular components (blebs) combined with synthetic liposomes and polymeric healing agents. Due to the transparent nature of PEDOT:PSS, the occurring biological phenomena were transduced in electronic output in real time, while the results were verified by state-of-the-art optical techniques.
Here, we demonstrate a general surface functionalization approach as a tool to introduce a variety of chemical groups on top of the PEDOT:PSS based electronic transducers. In the context of membrane-on-a-chip devices, our approach was used to tune the electrostatic interaction between the lipid bilayers and their electroactive supports. As a result of the increased affinity, membrane-on-a-chip devices with minimized and even eliminated synthetic component were made possible. Those devices were then used to provide mechanistic insight on biological events such as the binding and fusion of viruses. To demonstrate the capabilities of our platform, we monitored the interaction of a SARS-CoV-2 surrogate virus with a mouse-derived bilayer. When this coronavirus strain gets incubated and the virus fuses with the bilayer, a significant increase in impedance is observed, indicating that real-time electronic monitoring on such interactions is possible using our cell-free approach.
This simple platform has potential in assisting and re-directing drug discovery by providing real-time feedback early in the process, helping to reduce the high attrition rates during later stages when the costs are considerably higher.

Living cells can maintain their internal properties within a certain range, so-called homeostasis, e.g. intracellular pH. However, pH homeostasis has thus far not been looked at in the context of all the cellular physiological variables that are influenced by it, and the intertwined nature of these variables suggests that we need to so, to understand the homeostasis of even just one of them.
Here we build a simplified mathematical model of cellular electrophysiology. We show that bacteria use proton-ion antiporters to generate an out-of-equilibrium cytoplasmic membrane potential and so maintain the proton motive force (PMF) at the constant levels observed empirically. As a consequence, the strength of the PMF determines the range of extracellular pH over which the cell preserves a near-neutral cytoplasmic pH. We employ assays for time-course, single-cell, concurrent measurements of the PMF and pH and find agreement with our model prediction. We also demonstrate our efforts to measure the PMF in individual cells, but on thousands of them at the same time.
Our results suggest a new perspective on bacterial electrophysiology with cells maintaining their PMF and cytoplasmic pH by actively regulating the plasma membrane potential through changing activities of antiporters.

Photoactive organic semiconductors are envisioned as a novel class of materials able to transduce light into stimulating signals inside biological cells or tissue [1]. The direct interface between the semiconductor and the electrolyte gives rise to different, competing electrochemical phenomena such as the photofaradaic or the photocapacitive processes, depending whether the photogenerated charges get involved in redox processes or accumulate at an interface. A detailed understanding of such chemical and photo-induced interactions is necessary to develop and optimize future devices.
We addressed the problem in organic photoelectrodes considering both polymeric single-layer and organic molecular p/n junction thin-films [2]. Both are material systems that have been recently demonstrated to achieve the photomodulation of cardiac regeneration processes [3] or retinal neurons response [4].
By spectroscopic photovoltage and photocurrent measurements we gain insight into the energetics of the involved processes, while performing transient measurement we are able to identify the kinetics of light stimulated charge transfer processes. By combining these techniques with impedance spectroscopy, electric modelling and kinetic modelling, we identify the role of interfacial energy levels in the photoactivated processes and distinguish photocapacitive and photofaradaic contributions. The findings are further combined with nanoscale morphological investigations and Kelvin-Probe Force Microscopy leading to a quantitative interfacial energy diagram. We highlight how the energy diagram enables a comprehensive understanding of the photoelectrochemical reaction pathways and how the findings can be translated to predict the response of novel materials and devices, such as transducers based on floating semiconducting nanoparticles.

[1] J. Hopkins, L. Travaglini, A. Lauto, T. Cramer, B. Fraboni, J. Seidel, and D. Mawad, “Photoactive Organic Substrates for Cell Stimulation: Progress and Perspectives,” Adv. Mater. Technol., vol. 4, no. 5, pp. 1–10, 2019.
[2] T. Paltrinieri, L. Bondi, V. Derek, B. Fraboni, E. D. Glowacki, and T. Cramer, “Understanding Photocapacitive and Photofaradaic Processes in Organic Semiconductor Photoelectrodes for Optobioelectronics,” Adv. Funct. Mater., vol. 2010116, 2021.
[3] F. Lodola, V. Rosti, G. Tullii, A. Desii, L. Tapella, P. Catarsi, D. Lim, F. Moccia, and M. R. Antognazza, “Conjugated polymers optically regulate the fate of endothelial colony-forming cells,” Sci. Adv., vol. 5, no. 9, 2019.
[4] D. Rand, M. Jakesová, G. Lubin, I. Vebraite, M. David-Pur, et al., “Direct Electrical Neurostimulation with Organic Pigment Photocapacitors,” Adv. Mater., vol. 30, no. 25, pp. 1–11, 2018.

Supported lipid bilayers (SLBs) are a useful tool in the investigation of membrane proteins and their interactions at the single molecule level. Outer membrane vesicles (OMVs) can be combined with synthetic liposomes to form complete SLBs that contain the natural components of the bacterial outer membranes (Hsia et al., 2016). These systems show potential as novel antimicrobial screening platforms especially when integrated with bioelectronic devices. However, to date these hybrid SLBs have not been characterised at the molecular level. We employ correlative atomic force (AFM) with structured illumination microscopy (SIM) to gain detailed structural and functional information on those model systems (Ben-Sasson et al., 2021). SLBs containing OMVs derived from E. coli BL21(DE3) are studied to fully characterise the membrane protein, bacterial lipopolysaccharide and synthetic lipid components of the bilayers. We then increase the complexity of the system by engineering OMVs to contain proteins of interest, in this case a GFP binding nanobody-LppOmpA hybrid protein, and show that lipid patches that contain the overexpressed protein can be specifically identified and characterised. The relevance of these hybrid SLBs to antimicrobial screening applications is then demonstrated by SIM-AFM visualisation of known bacteria-targeting DNA nanostructures (Mela et al., 2020) binding selectively to bacterial components of the bilayers. Our study introduces correlative AFM-SIM as a powerful microscopy technique for the characterisation of lipid bilayers with multiple components and demonstrates the need for reproducible, programmable model systems for antimicrobial screening.

Heterogeneity is an omnipresent feature of biological populations, and the possibility to investigate it allows revealing important information that might be hidden otherwise. In this regard, it is fundamental to develop tools and techniques capable to investigate the specimens automatically and at high throughput, without affecting the quality of the analysis.
Among different 3D microscopy techniques, significant advantages in sample investigation are offered by light sheet fluorescence microscopy (LSFM). This method is based on a non-invasive optical sectioning approach that permits to obtain 3D information, by illuminating the sample plane by plane with a sheet of light. Standard implementations foresee the use of a cylindrical lens to focus the light in one direction only, while the excited the fluorescence is orthogonally collected by a microscope objective. The specimens are manually positioned in a sample chamber, aligned by the user in the optical system and automatically translated or rotated to allow multiple planes acquisition. This method permits fast image acquisition, low photobleaching and high signal to noise ratio. Nevertheless, the manual sample alignment requires up to several minutes, limiting the throughput of this technique.
The use of microfluidics to perform automatic sample delivery and translation could allow high throughput image acquisition of specimens diluted in a liquid suspension. Nevertheless, the integration of microfluidic channels in LSFM setups is not straightforward. This could introduce aberrations both to the excitation and collection path and it demands a very precise a stable alignment between the optical and fluidic system. Considering these constraints, we propose microfluidic based microscopes on a chip, integrating in a single substrate the optical elements necessary to create the light sheet such as optical waveguides and integrated cylindrical lenses as well as the microfluidic channel for automatic sample delivery. The integration of different components in a single platform guarantees the necessary alignment stability.
These microscopes on a chip have been fabricated in glass substrates by femtosecond laser micromachining (FLM) followed by chemical etching. Indeed, by focusing a pulsed laser beam into a transparent substrate, non-linear absorption processes occur and induce a permanent modification of the focal volume. This can lead to an increased etching selectivity or to a local increase of refractive index with respect to the pristine material, depending on the laser parameters. During sample irradiation, a 3D substrate translation permits to define the custom layout of the different components. Therefore, both microfluidic channels, integrated lenses as well as optical waveguides can be fabricated in the same substrate and within the same irradiation step by FLM.
Taking advantage of the versatility and capabilities of this technique, we propose different devices that have been adapted to the specificity of the specimens under investigation, such as cellular spheroids, Drosophila embryos and even single cells. Indeed, the sample channel size as well as the optical properties of the light sheets must be tailored considering the different size and shape of these samples in order to guarantee a good optical sectioning and a uniform illumination over the entire sample. Zemax simulations have been used to optimize the lens profile and to reduce both spherical and chromatic aberrations. Dual color and dual side illuminations have been demonstrated, with an image quality comparable of the one of standard LSFM, highlighting the capabilities of these microscopes on chip. In addition, the setup automation and the throughput of these devices have been optimized, proving an image acquisition rate of about 1 cell/second. Recently, by modifying the illumination path we have been able to further improve the image quality, proving a subcellular resolution.

Bacteria usually live in densely packed communities called biofilms, where the interactions between the bacteria give rise to complex properties. Quantitative analysis is very useful in understanding these properties. However, current biofilm culturing approaches impose various limitations. We developed a microfluidic approach for quantitative study of biofilms, which is universal and can be used to study biofilms of various bacterial species. To demonstrate the utility of this approach, we will present two examples, both of which revealed new biological insights. In the first example, we will explore the response of Escherichia coli biofilms to exogenous hydrogen peroxide. We found biofilms gained tolerance to H₂O₂, but their growth was slowed down due to the metabolic cost of maintaining the tolerance; However, under certain conditions, H₂O₂ can anti-intuitively boost growth. In the second example, we will show that in Pseudomonas aeruginosa biofilms, the extracellular matrix acts as a diffusion barrier for iron chelator, minimizing its loss into the environment and therefore potentially promoting the sharing of the chelator within the biofilm as a public good.

Exosomes are extracellular vesicles with phospholipid bilayer membrane that carry the encapsulated lipids, proteins, genetic materials and transmit in vivo. The exosomes those were secreted by specific cells and tissues show unique biological component and characteristics. Raman spectroscopy is a non-destructive and non-invasive technique that measuring the inelastic scattering of photons. It enables the analysis of the vibration mode of different biomolecules caused by monochromatic laser radiation. Distinguish and analysis of the Raman signal are difficult due to the low signal intensity. To overcome this problem, the surface-enhanced Raman spectroscopy (SERS) technique has been utilized. Gold and silver nanoparticles are typical used to amplify the signals in many systems include biosensing. A variety of SERS chips have been widely developed to obtain the signature spectra of the biomolecules. By using the SERS chip, liquid biological samples are dropped and dried before proceeding with the SERS technique. In this study, the SERS technique was employed in the colloid of the exosome-liquid mixture. Colloidal SERS offers the Raman spectra without the cell disruption and suitable for cells and tissues analysis. The exosomes purified from human embryonic kidney 293 (HEK 293) and rabies virus glycoprotein (RVG) were used. An only a small volume of exosomes (in a range of microliter) was decorated with silver NPs and diluted with 1 mL of phosphate-buffered saline (PBS). PBS is a water-based salt solution that has a pH similar to that of the human body. It is typically used to prevents the cells rupture and shrivels up. Interestingly, the SERS spectra were obtained with only 15 µL drop of the exosome-PBS mixture. The goal of this study is to develop a reliable and less complex point-of-care testing for practical usages with a handheld Raman spectrometer.

In this presentation, the colloidal SERS technique for detecting tissue-specific and nonspecific exosomes includes the concentration of the exosome-PBS mixture, the optimal volume of NPs, and the materials characterizations such as transmission electron microscopy (TEM), dynamic light scattering (DLS), and SERS spectra will be discussed.

Establishing close interactions between biological systems and synthetic materials is the key to form 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.^1,2 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.

A. M. Pappa et al Sci. Adv. 2018, 4 (6), eaat0911.
D. Ohayon et al Nat. Mater., 2020, 19, 456.

Membrane potential is known to be important for cross-membrane trafficking of ions and nutrients in all forms of life, but they are not limited to that. New discoveries show that electrophysiology plays a role in broader cellular and organism functions, ranging from antibiotic persistence to wound healing. Electrically induced membrane potential dynamics was recently shown to enable rapid detection of live bacteria with single-cell sensitivity, bridging an electro-bio hybrid system to diagnostics. It is still unclear, however, whether electrical stimulation can be used for the detection of live eukaryotic microbes. Here we show that proliferative and growth-inhibited budding yeast, Saccharomyces cerevisiae, exhibit distinct electrically induced membrane potential dynamics, enabling detection of proliferative eukaryotic cells from non-growing ones. When proliferative cells were stimulated by an electrical field, it induced hyperpolarization in plasma membrane. The same electrical stimulus, however, caused depolarisation when the cells were inhibited. The ability to detect not only prokaryotic, but also eukaryotic vital cells at single-cell level shows how promising electro-bio hybrid systems are as a tool for diagnostics. And the possibility to modulate membrane potential by applying an exogenous electrical stimulus sheds light on the potential use of electro-bio hybrid systems to regulate electrophysiology-dependent cellular functions, paving the path for future engineering of environment responding living-materials.

Electronic components that bridge the biotic-abiotic interface will have vast implications for both studying and harnessing the activity of living cells. While much ongoing research focuses on applying traditional rigid electronics to biology, an alternative is to discover bioelectronic solutions that life itself evolved to interact with the abiotic world. Towards realizing this vision, recent studies at the interface of microbiology, electrochemistry, and physics have uncovered metalloprotein electron conduits and nanowires that electronically link bacteria to extracellular surfaces ranging from environmental minerals to solid-state electrodes. Since this extracellular electron transport naturally evolved to interact with external surfaces, a fundamental understanding has special implications for new bioelectrochemical technologies and living electronics that harness the advantages of microbes in detecting external signals or hosting synthetic genetic circuits.

This talk will describe our group's recent progress in understanding extracellular electron transport at multiple length scales, from the biophysics of individual multiheme cytochromes to the electrophysiology of whole bacteria and multicellular communities ranging from biofilms to cable bacteria. Using single molecule tracking and stochastic simulations of cell surface multiheme cytochromes in the metal-reducing bacterium Shewanella oneidensis MR-1, we describe how the interplay of cytochrome dynamics and electron hopping can give rise to long-distance electron conduction along bacterial membrane surfaces. In addition, we describe strategies to characterize and harness the electrochemical activity, spin filtering, and conduction properties of bacterial electron conduits in both synthetic structures and living biofilm materials.

Electrical stimulation therapies are increasingly useful for nervous system disorders but even state of the art (metal) electrode materials have limitations, such as unwanted production of heat, free radical species, pH changes and associated tissue necrosis. Implanted electrodes also may need to be hard wired to a power source, though indirect electrostimulation is possible by creating an electrical gradient wirelessly through generation of dipoles at the borders of implanted materials by bipolar electrochemistry. Improved understanding of how neurons and materials interact to influence nerve growth would be valuable for future restorative electro-therapies.
By delivering a voltage gradient through the culture medium in vitro we have explored neuron growth on insulating materials, such as glass or tissue culture plastic, and compared it to conducting materials, such as gold or platinum and also to conducting intercalation materials. Materials were prepared as thin coatings to permit optical transparency and to allow observation of growth dynamics. Neurons from the developing amphibian spinal cord were used, mainly because, unlike mammalian neurons, they do not require any adhesion factors between the cell and the underlying substrate.
Neurons grew faster and more directly toward the external driving cathode when plated onto tissue culture plastic (insulating material) and, when the stimulation was within a safe window, they turned to grow toward the cathode on gold. Differences were apparent between conducting and mixed conducting intercalation materials, with neurons grown on IrOx favouring speed of growth (but no directional growth) and those on PEDOT-SS based materials favoring turning towards the external driving cathode (but not faster growth).
The data suggest that indirect electrical stimulation of neurons can be tuned to achieve specific growth responses by designing implant materials with specific bipolar electrochemical traits, even in challenging situations where transparency is required. This has clear potential to for designing neural prostheses or neural repair strategies but the concepts could also be applied to other wound healing and regeneration situations.

One of the ultimate goals of bioelectronics is an ability to electronically access all that biology has to offer - to control and record the myriad of native and engineered biological processes inside of cells and organisms. This could be enabled by seamless and dynamic bidirectional communication between electronic and biological components. As a step towards realizing these capabilities, electrogenetics offers tools to use electrical stimuli to modulate engineered gene expression. Electronic control of the oxidation state of redox molecules modulates redox-sensing proteins and gene expression from the promoters they activate. We have co-opted the SoxR and OxyR redox-sensing proteins from Escherichia coli in two distinct electrogenetic systems. These allow us to electronically control the expression of a number of engineered genes and behaviors in various environments and configurations. We developed a multi-functional microbial community, the BioLAN, which reliably facilitates on-demand bioelectronics communication and electrogenetic control of a programmed biological task. The BioLAN also provides electronically-recordable cell-based reporting to allow for feedback control. The electrogenetic systems presented here can be incorporated into genetic circuits and logic gates alongside chemical and light-driven components. They present a melding of synthetic biology and bioelectronics towards building seamlessly-integrated bio-hybrid devices.

I will present our recent efforts to understand and manipulate the membrane potential of bacteria that make up biofilm communities. In particular, I will talk about our efforts to encode memory in space and time within biofilms using the membrane potential of bacteria. This work can set the stage to utilize biofilms for performing computations. I will end by showing some new results regarding our ability to control biofilms.