We developed an electrobiofabrication methodology that assembles well‐defined cell/gel formations directly onto electrodes. For this, we oxidatively crosslinked terminal thiols of a 4‐arm thiolated polyethylene glycol (PEG) by the purposeful addition of a ferrocene redox mediator to a PEG/cell assembly solution and the application of an oxidizing charge to an electrode. Because the resulting disulfide bonds are created near the electrode, the crosslinked hydrogel assembly is defined by the electrode dimensions and the time over which the oxidative potential is applied. Results indicate a strong positive correlation between the mediator concentration, the delivered oxidative charge, the number density of cells in the assembly solution and the subsequent gel thickness and density. In all cases tested, the viability of the assembled cells (E. coli bacteria) was near 100%. We further demonstrated a gravity‐mediated layering methodology to create spatially defined interfaces, as well as electroassembly onto various conductive materials of nearly arbitrary shape. These results represent a means for electronic or “programed” assembly of cell laden hydrogels, enabling further study of cell–cell interactions, cell‐device interactions, biosensing, device ⇔ bio communication, and several applications such as electrogenetics wherein cell genetic circuits are actuated by application of electrical potentials using a redox‐enabled communication modality.

The rapid development of and the growing need for recapitulating desired tissue geometries in 3D on Organ on-a-Chip (OOC) and printed microscale scaffolds has revealed new challenges on cell seeding processes. Initially, researchers had made significant progress on utilizing techniques (e.g. "Two-Photon Direct Laser Writing (DLW)") to create sophisticated micro scaffolds with critical features such as micropores, and interweaving vessels. However, the basic cell seeding strategy has not changed the cell proliferation on the scaffolds and the formation of desired morphologies typically relies heavily on cell self-assembly and long incubation times. That is, for conventional two-dimensional models, cells naturally settle on the prepared membranes, while for advanced 3D models, there is often a desire for cells to occupy certain regions while excluding other critical locations. To provide a new methodology to address these issues, we leverage precise pressure manipulation and heterogeneous interfaces across building materials to achieve rapid cell assembly onto desired locations of complex micro 3D printed scaffolds. Firstly, we created two testing models, including scaffolds with micropores and micro trusses with low coverages. Preliminary experimental results for the pressure-driven cell assembly shows pore coverage over 90% and when needed, instantaneous release on porous scaffolds within 1.5 minute. The hydrogel with the cell mimics deployment onto the micro truss also builds a reliable hybrid structure that suits for cell anchoring within 5 minutes. Together, this work serves as a fundamental proof-of-principle for rapidl assembly of cells on future microphysiological mimics where complicated features arc needed for diverse OOC applications.

Inflammatory bowel diseases (IBD) affect over 6 million people globally and current treatments achieve only 10-20% rates of durable disease remission. Bacterial extracellular vesicles (BEVs) from probiotic lactic acid bacteria (LAB) are a promising novel therapeutic with mechanisms holding potential to drive increased rates of durable disease remission, including immunomodulation and intestinal epithelial tissue repair. However, translation of these cell-secreted nanovesicles is limited by long standing biomanufacturing hurdles, especially low production yields due to low biogenesis rates from cells. Here, Lactiplantibacillus plantarum is identified as a candidate LAB producing BEVs effective in treating acute dextran sulfate sodium (DSS)-induced murine colitis with greater efficacy than BEVs from probiotic Escherichia coli Nissle 1917. Genetic engineering of L. plantarum to create a hypervesiculating strain via inducible expression of a peptidoglycan-modifying enzyme is shown to enable a 66-fold increase in BEV productivity. Finally, hypervesiculating L. plantarum BEVs are confirmed to be therapeutically effective in the acute DSS mouse model of colitis, with superior reduction of mucosal tissue damage compared to live L. plantarum cells. These findings demonstrate that BEVs from genetically engineered hypervesiculating strain of L. plantarum are a promising preclinical therapeutic candidate for IBD that overcomes historical biomanufacturing limitations of BEV therapeutics.

Microvessels (e.g., capillaries) are ubiquitous throughout human anatomy, yet recreating their three-dimensional (3D) microfluidic and architectural sophistication at biologically accurate length scales has remained a critical challenge. To overcome this barrier, here we report a hybrid additive manufacturing-or "3D printing"-strategy in which "Two-Photon Direct Laser Writing (DLW)" is used to nanoprint microvessels of arbitrary design directly atop "Liquid-Crystal Display (LCD)" 3D-printed microfluidic chips. Fabrication results indicated effective production of 100 μm-diameter 3D polydimethylsiloxane (PDMS) microfluidic vessels with 5 μm-thick walls-featuring arrays of pre-designed 5 μm-diameter micropores-as well as three discrete spiralled, intertwined microvessels. Experimental results with MDA-MB-231 epithelial breast cancer cells revealed the ability for the 3D PDMS microvessels to support cell culture. In combination, these results suggest that the presented strategy for 3D nanoprinting PDMS microvessels with custom-designed architectures and microporosity offers a promising pathway to enable new classes of "organ-on-a-chip (OOC)" systems for wide-ranging biomedical applications.

Redox, a native modality in biology involving the flow of electrons, energy, and information, is used for energy-harvesting, biosynthesis, immune-defense, and signaling. Because electrons (in contrast to protons) are not soluble in the medium, electron-flow through the redox modality occurs through redox reactions that are sometimes organized into pathways and networks (e.g., redox interactomes). Redox is also accessible to electrochemistry, which enables electrodes to receive and transmit electrons to exchange energy and information with biology. In this Perspective, efforts to develop electrochemistry as a tool for redox-based bio-information processing: to interconvert redox-based molecular attributes into interpretable electronic signals, are described. Using a series of Case Studies, how the information-content of the measurements can be enriched using: diffusible mediators; tuned electrical input sequences; and cross-modal measurements (e.g., electrical plus spectral), is shown. Also, theory-guided feature engineering approaches to compress the information in the electronic signals into quantitative metrics (i.e., features) that can serve as correlating variables for pattern recognition by data-driven analysis are described. Finally, how redox provides a modality for electrogenetic actuation is illustrated. It is suggested that electrochemistry's capabilities to provide real-time, low-cost, and high-content data in an electronic format allow the feedback-control needed for autonomous learning and deployable sensing/actuation.

Redox provides unique opportunities for interconverting molecular/biological information into electronic signals. Here, the fabrication of a 3D-printed multiwell device that can be interfaced into existing laboratory instruments (e.g., well-plate readers and microscopes) to enable advanced redox-based spectral and electrochemical capabilities is reported. In the first application, mediated probing is used as a soft sensing method for biomanufacturing: it is shown that electrochemical signal metrics can discern intact mAbs from partially reduced mAb variants (fragmentation), and that these near-real-time electrical measurements correlate to off-line chemical analysis. In the second application, operando spectroelectrochemical measurements are used to characterize a redox-active catechol-based hydrogel film: it is shown that electron transfer into/from the film correlates to the molecular switching of the film's redox state with the film's absorbance increasing upon oxidation and the film's fluorescence increasing upon reduction. In the final example, a synthetic biofilm containing redox-responsive E. coli is electro-assembled: it is shown that gene expression can be induced under reducing conditions (via reductive H2O2 generation) or oxidative conditions (via oxidation of a phenolic redox-signaling molecule). Overall, this work demonstrates that 3D printing allows the fabrication of bespoke electrochemical devices that can accelerate the understanding of redox-based phenomena in biology and enable the detection/characterization redox activities in technology.

It has been long appreciated that expression of the Yersinia type-III secretion system (T3SS) in culture is associated with growth arrest. Here we sought to understand whether T3SS expression is sufficient to trigger loss of exponential phase markers, and utilized a fluorescent reporter for ribosomal protein expression to detect changes in bacterial growth state. Using a fluorescent transcriptional reporter with the rpsJ/S10 promoter fused to a destabilized gfp variant, we confirmed reporter expression significantly increases in exponential phase and decreases as cells transition to stationary phase. In a mouse model of systemic Y. pseudotuberculosis infection, we found multiple subsets of bacterial cells in the mouse spleen, including cells with high T3SS and low S10 expression and cells with high expression of both markers. In bacterial media, growth inhibition with T3SS induction and a reduction in S10 expression were observed, but a significant proportion of cells retained high expression of both T3SS and S10. Paradoxically, while loss of T3SS expression rescued growth, lower S10 expression was detected, again indicating bacteria can express both markers simultaneously. In media, bacteria grow planktonically as individual cells, while in mouse tissues, bacteria form clustered extracellular communities. We utilized droplet-based microfluidics to encapsulate bacteria in spherical agarose droplets and model clustered growth, and observed high expression of T3SS without an impact on S10 levels. Finally, we show that T3SS expression is sufficient to promote antibiotic tolerance, but surviving bacteria in a gentamicin treatment mouse model specifically express low S10. Collectively, these data indicate that the growth arrest associated with T3SS induction can reduce antibiotic susceptibility, but cells surviving antibiotic treatment display lower levels of the exponential phase marker, S10.

Introduction: Molecular communication is the transfer of information encoded by molecular structure and activity. We examine molecular communication within bacterial consortia as cells with diverse biosynthetic capabilities can be assembled for enhanced function. Their coordination, both in terms of engineered genetic circuits within individual cells as well as their population-scale functions, is needed to ensure robust performance. We have suggested that “electrogenetics,” the use of electronics to activate specific genetic circuits, is a means by which electronic devices can mediate molecular communication, ultimately enabling programmable control.Methods: Here, we have developed a graphical network model for dynamically assessing electronic and molecular signal propagation schemes wherein nodes represent individual cells, and their edges represent communication channels by which signaling molecules are transferred. We utilize graph properties such as edge dynamics and graph topology to interrogate the signaling dynamics of specific engineered bacterial consortia.Results: We were able to recapitulate previous experimental systems with our model. In addition, we found that networks with more distinct subpopulations (high network modularity) propagated signals more slowly than randomized networks, while strategic arrangement of subpopulations with respect to the inducer source (an electrode) can increase signal output and outperform otherwise homogeneous networks.Discussion: We developed this model to better understand our previous experimental results, but also to enable future designs wherein subpopulation composition, genetic circuits, and spatial configurations can be varied to tune performance. We suggest that this work may provide insight into the signaling which occurs in synthetically assembled systems as well as native microbial communities.

Melanins have complex structures, difficult-to-characterize properties, and poorly understood biological functions. Electrochemical methods are revealing how melanin's redox-state molecular-switching is coupled to its electron-transfer activities.

The proline amino acid and prolyl residues of peptides/proteins confer unique biological and biochemical properties that motivates the development of proline-selective analysis. The study focuses on one specific class of problem, the detection of single amino acid variants involving proline, and reports a Pro-selective electrochemiluminescence (ECL) method. To develop this method, the A1-/A2- variants of milk's β-casein protein are investigated because it is a well-established example and abundant samples are readily available. Specifically, β-casein has 209 amino acids with 34 (or 35) proline residues: the A1-variant has a Pro-to-His substitution at position 67 (relative to the A2 variant). The study shows that proline's strong luminescence allows the generic discrimination of: Pro from other amino acids; an A2-oligopeptide from an A1-oligopeptide; the A2-β-casein variant from the A1-variant; and commercially-available A2 milks from A1-containing regular milks. The evidence indicates that luminescence depends on proline content and accessibility, as well as signal quenching. Compared to conventional immunoassays, the ECL method is simple, rapid, and inexpensive. Further, the ECL-method is Pro-selective (vs molecularly-selective like typical immunoassays) which should make it broadly useful for studying the role of proline in biology and especially useful for tracking the digestion of proline-rich proteins in the diet.

Electronic communication in natural systems makes use, inter alia, of molecular transmission, where electron transfer occurs within networks of redox reactions, which play a vital role in many physiological systems. In view of the limited understanding of redox signaling, we developed an approach and an electrochemical-optical labon-a-chip to observe cellular responses in localized redox environments. The developed fluidic micro-system uses electrogenetic bacteria in which a cellular response is activated to electrically and chemically induced stimulations. Specifically, controlled environments for the cells are created by using microelectrodes to generate spatiotemporal redox gradients. The in-situ cellular responses at both single-cell and population levels are monitored by optical microscopy. The elicited electrogenetic fluorescence intensities after 210 min in response to electrochemical and chemical activation were 1.3 x 108 +/- 0.30 x 108 arbitrary units (A.U.) and 1.2 x 108 +/- 0.30 x 108 A.U. per cell population, respectively, and 1.05 +/- 0.01 A.U. and 1.05 +/- 0.01 A.U. per-cell, respectively. We demonstrated that redox molecules' mass transfer between the electrode and cells - and not the applied electrical field - activated the electrogenetic cells. Specifically, we found an oriented amplified electrogenetic response on the charged electrodes' downstream side, which was determined by the location of the stimulating electrodes and the flow profile. We then focused on the cellular responses and observed distinct subpopulations that were attributed to electrochemical rather than chemical stimulation, with the distance between the cells and the stimulating electrode being the main determinant. These observations provide a comprehensive understanding of the mechanisms by which diffusible redox mediators serve as electron shuttles, imposing context and activating electrogenetic responses.

Protein function relies on sequence, folding and post-translational modification and molecular measurements are commonly used to reveal these structural features. Here, we report an alternative approach that represents these molecular features as readily measurable electronic patterns and validate this experimental approach by detecting structural perturbations commonly encountered during protein biomanufacturing. We studied a monoclonal antibody standard (from the National Institute of Standards and Technology) and focused on the electronic detection of variants that have undergone interchain disulfide bond reduction and methionine oxidation. Electronic detection of these structural perturbations is based on mediated electrochemical probing (MEP) that discerns patterns associated with the antibody’s mediator-accessible redox activity. We demonstrate that MEP can rapidly (within minutes) and quantitatively detect alterations in the antibody’s structural features and produce robust electronic signals that could enable monitoring of biomanufacturing processes. The ability to transduce information regarding a protein’s structural perturbations into a more convenient electronic domain offers opportunities to apply the power of microelectronics and real-time data analytics to chemical and biological analysis. A method called mediated electrochemical probing (MEP) was developed, which enables the rapid conversion of information about structural perturbations of proteins into convenient, readily accessible electronic data. MEP was used to detect structural variants of a monoclonal antibody by discerning patterns associated with the antibody’s mediator-accessible redox activity.

Redox is a unique, programmable modality capable of bridging communication between biology and electronics. Previous studies have shown that the E. coli redox-responsive OxyRS regulon can be re-wired to accept electrochemically generated hydrogen peroxide (H 2 O 2 ) as an inducer of gene expression. Here we report that the redox-active phenolic plant signaling molecule acetosyringone (AS) can also induce gene expression from the OxyRS regulon. AS must be oxidized, however, as the reduced state present under normal conditions cannot induce gene expression. Thus, AS serves as a “pro-signaling molecule” that can be activated by its oxidation - in our case by application of oxidizing potential to an electrode. We show that the OxyRS regulon is not induced electrochemically if the imposed electrode potential is in the mid-physiological range. Electronically sliding the applied potential to either oxidative or reductive extremes induces this regulon but through different mechanisms: reduction of O 2 to form H 2 O 2 or oxidation of AS. Fundamentally, this work reinforces the emerging concept that redox signaling depends more on molecular activities than molecular structure. From an applications perspective, the creation of an electronically programmed “pro-signal” dramatically expands the toolbox for electronic control of biological responses in microbes, including in complex environments, cell-based materials, and biomanufacturing.

Electrode-imposed electronic inputs can generate various cues that can control the emergence of hierarchical structure and confer function to hydrogel systems. Here we describe three such top-down cues. Electrolytic reactions can create pH cues that can induce the electrodeposition of pH-responsive self-assembling polymers (e.g., chitosan and alginate). The electric field provides a long-range cue that can induce polymer chains to migrate toward (or away from) the electrode and can align the polymer chains within the assembling hydrogel network (e.g., collagen). The electrochemical generation of diffusible oxidants provides a molecular cue that can induce oxidative assembly - typically through the formation of covalent bonds (e.g., disulfide bonds). Here, we review recent results on the use of these three cues for the electrofabrication of hydrogels and we illustrate how complementary capabilities from biotechnology allow the creation of functional hydrogel systems. Overall, we envision that electro-bio-fabrication could emerge as a scalable additive manufacturing method as well as a flexible approach for distributed manufacturing in public maker spaces.

Microphysiological systems-also known as "organ-on-a-chip (OOC)" systems-hold considerable promise for applications including drug screening, disease modeling, and personalized medicine. A critical barrier to OOC efficacy, however, stems from manufacturing challenges that hinder the accurate recreation of 3D architectures and material properties of in vivo organ systems. To provide a new pathway to address these issues, here we leverage "Two-Photon Direct Laser Writing (DLW)" to 3D print physiologically relevant polydimethylsiloxane (PDMS) microvessels directly atop 3D microfluidic chips-fabricated via the "Vat Photopolymerization (VPP)" technique, "Liquid-Crystal Display (LCD)" 3D printing. Fabrication results revealed effective production of both interweaving and independent 3D microfluidic vessels with inner diameters (IDs) and wall thicknesses ranging from 80-100 mu m and 5-10 mu m, respectively, as well as pre-designed (i.e., as-printed) micropores with 5 mu m diameters. Preliminary experimental results for MDA-MB-231 cells seeded within the porous microvessels revealed that the 3D PDMS system supported cell viability. In addition, pressure-vacuum experiments revealed that the permeation effects could be tuned to regulate the microenvironmental conditions internal and external to the porous microvessels. In combination, this work serves as a fundamental proof of principle for establishing entirely new classes of 3D microphysiological systems for diverse OOC applications.

One-third of people with schizophrenia have elevated levels of anti-gliadin antibodies (AGA IgG). A 5-week randomized double-blind pilot study was performed in 2014-2017 in an inpatient setting to test the effect of a gluten-free diet (GFD) on participants with schizophrenia or schizoaffective disorder who also had elevated AGA IgG (>= 20 U) but were negative for celiac disease. This earlier pilot study reported that the GFD-group showed improved gastrointestinal and psychiatric symptoms, and also improvements in TNF-alpha and the inflammatory cytokine IL-23. Here, we performed measurements of these banked plasma samples to detect levels of oxidative stress (OxSt) using a recently developed iridium (Ir)-reducing capacity assay. Triplicate measurements of these samples showed an Intraclass Correlation Coefficient of 0.84 which indicates good reproducibility. Further, a comparison of the OxSt measurements at the baseline and 5-week end-point for this small sample size shows that the GFD-group (N = 7) had lowered OxSt levels compared to the gluten-containing diet group (GCD; N = 9; p = 0.05). Finally, we showed that improvements in OxSt over these 5 weeks were correlated to improvements in gastrointestinal (r = +0.64, p = 0.0073) and psychiatric (r = +0.52, p = 0.039) symptoms. Also, we showed a possible association between the decrease in OxSt and the lowered levels of IL-23 (r = +0.44, p = 0.087), although without statistical significance. Thus, the Ir-reducing capacity assay provides a simple, objective measure of OxSt with the results providing further evidence that inflammation, redox dysregulation and OxSt may mediate interactions between the gut and brain.

We report the integration of 3D printing, electrobiofabrication, and protein engineering to create a device that enables near real-time analysis of monoclonal antibody (mAb) titer and quality. 3D printing was used to create the macroscale architecture that can control fluidic contact of a sample with multiple electrodes for replicate measurements. An analysis "chip" was configured as a "snap-in" module for connecting to a 3D printed housing containing fluidic and electronic communication systems. Electrobiofabrication was used to functionalize each electrode by the assembly of a hydrogel interface containing biomolecular recognition and capture proteins. Specifically, an electrochemical thiol oxidation is used to assemble a thiolated polyethylene glycol hydrogel, that in turn is covalently coupled to either a cysteine-tagged protein G that binds the antibody's Fc region or a lectin that binds the glycans of target mAb analytes. We first show the design, assembly, and testing of the hardware device. Then, we show the transition of a step-by-step sensing methodology (e.g., mix, incubate, wash, mix, incubate, wash, measure) into the current method where functionalization, antibody capture, and assessment are performed in situ and in parallel channels. Both titer and glycan analyses were found to be linear with antibody concentration (to 0.2 mg/L). We further found the interfaces could be reused with remarkably similar results. Because the interface assembly and use are simple, rapid, and robust, we suggest this assessment methodology will be widely applicable, including for other biomolecular process development and manufacturing environments.

In biopharmaceutical manufacturing, current methods of quality control utilize intensive analytical techniques that provide detailed information but consist of long processing times. Timelines for yield quantification and product quality assessment can sometimes take days, missing the window to correct for production errors. The field would benefit from rapid detection of products for quality control and optimization. By leveraging redox chemistry of bioactive compounds, recent developments in electroanalytical techniques aim to fill these gaps. However, standardized metrics and data analysis for the interpretation of these measurements are necessary for widespread application. With the high throughput capabilities of electroanalytical techniques, we suggest that a data pipeline for classification and quantification of molecular products utilizing machine learning models would greatly enhance the applicability of these methods. We demonstrate feasibility of appropriate dataset generation for amino acids and proteins measured using cyclic voltammetry. We then develop machine learning models for classification and quantification of samples based on voltammograms alone. The pipeline outlined in this work offers a robust and simple method for delineating biological electroanalytical data, expanding application potential for diagnostics in both manufacturing and clinical settings.

The toxicity of paraquat is believed to involve a redox-cycling mechanism that can disrupt cellular redox homeostasis and, also, generate damaging free radicals. It has been suggested that for cases of severe paraquat poisoning the administration of ascorbate (i.e., vitamin C) can confer benefit by quenching the paraquat free radical (PQ+·). Here, we used an electrochemical approach that abstracts-away many of the (bio)chemical complexities and isolates the redox-interactions between paraquat and ascorbate. Specifically, we used a series of experiments that coupled electrochemical measurements of electron flow with optical measurements of paraquat's redox-state switching. Our results demonstrate that the reduced absorbate cannot quench the PQ+·-radical because they are both reductants. However, oxidation of ascorbate does allow PQ+·-radical scavenging. More broadly, we believe this study demonstrates the potential for developing electrochemical approaches to complement existing experimental methods in redox biology.