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Abstract 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. 

Abstract 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,operandospectroelectrochemical 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‐responsiveE. coliis electro‐assembled: it is shown that gene expression can be induced under reducing conditions (via reductive H₂O₂generation) 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. 

Abstract Bacterial extracellular vesicles (BEVs), including outer membrane vesicles, have emerged as a promising new class of vaccines and therapeutics to treat cancer and inflammatory diseases, among other applications. However, clinical translation of BEVs is hindered by a current lack of scalable and efficient purification methods. Here, we address downstream BEV biomanufacturing limitations by developing a method for orthogonal size‐ and charge‐based BEV enrichment using tangential flow filtration (TFF) in tandem with high performance anion exchange chromatography (HPAEC). The data show that size‐based separation coisolated protein contaminants, whereas size‐based TFF with charged‐based HPAEC dramatically improved purity of BEVs produced by probiotic Gram‐negativeEscherichia coliand Gram‐positive lactic acid bacteria (LAB).Escherichia coliBEV purity was quantified using established biochemical markers while improved LAB BEV purity was assessed via observed potentiation of anti‐inflammatory bioactivity. Overall, this work establishes orthogonal TFF + HPAEC as a scalable and efficient method for BEV purification that holds promise for future large‐scale biomanufacturing of therapeutic BEV products. 

Abstract β‐galactosidase (β‐gal) is one of the most prevalent markers of gene expression. Its activity can be monitored via optical and fluorescence microscopy, electrochemistry, and many other ways after slight modification using protein engineering. Here, we have constructed a chimeric version that incorporates a streptococcal protein G domain at the N‐terminus of β‐gal that binds immunoglobins, namely IgG. This protein G: β‐galactosidase fusion enables β‐gal‐based spectrophotometric and electrochemical measurements of IgG. Moreover, our results show linearity over an industrially relevant range. We demonstrate applicability with rapid spectroelectrochemical detection of IgG in several formats including using an electrochemical sensing interface that is rapidly assembled directly onto electrodes for incorporation into biohybrid devices. The fusion protein enables sensitive, linear, and rapid responses, and in our case, makes IgG measurements quite robust and simple, expanding the molecular diagnostics toolkit for biological measurement. 

Abstract Electronic materials that allow the controlled flow of electrons in aqueous media are required for emerging applications that require biocompatibility, safety, and/or sustainability. Here, a composite hydrogel film composed of graphene and catechol is electrofabricated, and that this composite offers synergistic properties is reported. Graphene confers metal‐like conductivity and enables charge‐storage through an electrical double layer mechanism. Catechol confers redox‐activity and enables charge‐storage through a redox mechanism. Importantly, there are two functional populations of catechols: conducting‐catechols (presumably in intimate contact with graphene) allow direct electron‐transfer; and non‐conducting‐catechols (presumably physically separated from graphene) require diffusible mediators to enable electron‐transfer. Using a variety of spectroelectrochemical measurements, that the capacity of the composite for charge‐storage increases in proportion to the extent by which the catechol‐groups can undergo redox‐state switching is demonstrated. To illustrate the broad relevance of this work, how the redox‐state switching can be related to both the charge storage of energy materials and the memory of molecular electronic materials is discussed. The authors believe this work is significant because it demonstrates that: conducting and redox‐active components enable distinctly different mechanisms for charge‐storage and electron‐transfer; these components act synergistically; and mediators provide unique opportunities to extend the capabilities of electronic materials. 

In addition to engineering new pathways for synthesis, synthetic biologists rewire cells to carry out “programmable” functions, an example being the creation of wound‐healing probiotics. Engineering regulatory circuits and synthetic machinery, however, can be deleterious to cell function, particularly if the “metabolic burden” is significant. Here, a synthetic regulatory circuit previously constructed to directEscherichiacolito swim toward hydrogen peroxide, a signal of wound generation, was shown to work even with coexpression of antibiotic resistance genes and genes associated with lactose utilization. We found, however, that cotransformation with a second vector constitutively expressing GFP (as a marker) and additionally conferring resistance to kanamycin and tetracycline resulted in slower velocity (Δ~6 μm/s) and dramatically reduced growth rate (Δ > 50%). The additional vector did not, however, alter the run‐and‐tumble ratio or directional characteristics of H₂O₂–dependent motility. The main impact of this additional burden was limited to slowing cell velocity and growth, suggesting that reprogrammed cell motility by minimally altering native regulatory circuits can be maintained even when extraneous burden is placed on the host cell. © 2019 American Institute of Chemical EngineersBiotechnol. Prog., 35: e2778, 2019. 

Abstract Emerging research indicates that biology routinely uses diffusible redox‐active molecules to mediate communication that can span biological systems (e.g., nervous and immune) and even kingdoms (e.g., a microbiome and its plant/animal host). This redox modality also provides new opportunities to create interactive materials that can communicate with living systems. Here, it is reported that the fabrication of a redox‐active hydrogel film can autonomously synthesize a H₂O₂signaling molecule for communication with a bacterial population. Specifically, a catechol‐conjugated/crosslinked 4‐armed thiolated poly(ethylene glycol) hydrogel film is electrochemically fabricated in which the added catechol moieties confer redox activity: the film can accept electrons from biological reductants (e.g., ascorbate) and donate electrons to O₂to generate H₂O₂. Electron‐transfer from anEscherichia coliculture poises this film to generate the H₂O₂signaling molecule that can induce bacterial gene expression from a redox‐responsive operon. Overall, this work demonstrates that catecholic materials can participate in redox‐based interactions that elicit specific biological responses, and also suggests the possibility that natural phenolics may be a ubiquitous biological example of interactive materials. 

Abstract Process conditions established during the development and manufacture of recombinant protein therapeutics dramatically impacts their quality and clinical efficacy. Technologies that enable rapid assessment of product quality are critically important. Here, we describe the development of sensor interfaces that directly connect to electronics and enable near real‐time assessment of antibody titer and N‐linked galactosylation. We make use of a spatially resolved electroassembled thiolated polyethylene glycol hydrogel that enables electroactivated disulfide linkages. For titer assessment, we constructed a cysteinylated protein G that can be linked to the thiolated hydrogel allowing for robust capture and assessment of antibody concentration. For detecting galactosylation, the hydrogel is linked with thiolated sugars and their corresponding lectins, which enables antibody capture based on glycan pattern. Importantly, we demonstrate linear assessment of total antibody concentration over an industrially relevant range and the selective capture and quantification of antibodies with terminal β‐galactose glycans. We also show that the interfaces can be reused after surface regeneration using a low pH buffer. Our functionalized interfaces offer advantages in their simplicity, rapid assembly, connectivity to electronics, and reusability. As they assemble directly onto electrodes that also serve as I/O registers, we envision incorporation into diagnostic platforms including those in manufacturing settings. 

Abstract Biology uses diffusible oxidants to perform functions that range from signaling to matrix assembly, and these oxidation chemistries offer surprising selectivities. Here, it is reported that mediated electrochemistry can access the richness of such oxidation chemistries. Specifically, electrode‐imposed voltage inputs are used to locally generate oxidized mediators that can diffuse into polymer solutions and induce the formation of covalent bonds for the deposition and functionalization of hydrogels at the electrode surface. Depending on the mediator's redox potential (E⁰), it is possible to “gate” the voltage inputs to target specific residues (e.g., thiols or amines) and oxidation chemistries. Further, mediators of varyingE⁰offer different reactivities and thus allow control of reaction‐diffusion rates to modulate the hydrogel's crosslink density and mechanical properties. Importantly, this mediated oxidation can be performed under physiologically relevant conditions to preserve labile biological functionalities (e.g., cell viability and protein function). Finally, it is demonstrated that protein fusion tags can be engineered to have “targetable” amino acid residues that enable protein function to be oxidatively conjugated to electrodeposited hydrogels. In summary, mediated electrochemistry can engage orthogonal oxidation chemistries to create functionalized matrices and thus mediated electrochemistry should add important capabilities to the electrofabrication toolbox. 

Abstract Reduction–oxidation (redox) reactions provide a distinct modality for biological communication that is fundamentally different from the more‐familiar ion‐based electrical modality. Biology uses these two modalities for communication through different systems (immune versus nervous), and uses different mechanisms to control the flow of the charge carriers: the flow of soluble ions is controlled using structural barriers (i.e., membranes) and gates (e.g., membrane‐spanning protein channels), while the flow of insoluble electrons is controlled using redox‐reaction networks. Here, a simple electrochemical approach to pattern catechols onto a flexible polysaccharide hydrogel is reported and it is demonstrated that the patterned catechol regions serve as nodes for the mediated flow of electrons through redox reactions. Electron flow through this node involves the switching of binary redox states (oxidized and reduced) and this node's redox state can be detected (i.e., “read”) by passively observing its optical absorbance, or actively switching its redox‐state electrochemically. Further, this catechol node can be switched through biological mechanisms, and this enables the fabricated catechol node to be embedded within biochemical redox reaction networks to facilitate the spanning of bio‐electronic communication. Thus, it is envisioned that catechols can emerge as a molecular equivalent to a transistor for miniaturize‐able, deployable and sustainable redox‐linked bioelectronics.