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Wastewater treatment using aerobic granular sludge has gained increasing interest due to its advantages compared to conventional activated sludge. The technology allows simultaneous removal of organic carbon, nitrogen, and phosphorus in a single reactor system and is independent of space-intensive settling tanks. However, due to the microscale, an analysis of processes and microbial population along the radius of granules is challenging. Here, we introduce a model system for aerobic granular sludge on a small scale by using a machine-assisted microfluidic cultivation platform. With an implemented logic module that controls solenoid valves, we realized alternating oxic hunger and anoxic feeding phases for the biofilms growing within. Sampling during ongoing anoxic cultivation directly from the cultivation channel was achieved with a robotic sampling device. Analysis of the biofilms was conducted using optical coherence tomography, fluorescence in situ hybridization, and amplicon sequencing. Using this setup, it was possible to significantly enrich the percentage of polyphosphate-accumulating organisms (PAO) belonging to the family Rhodocyclaceae in the community compared to the starting inoculum. With the aid of this miniature model system, it is now possible to investigate the influence of a multitude of process parameters in a highly parallel way to understand and efficiently optimize aerobic granular sludge-based wastewater treatment systems.Key points• Development of a microfluidic model to study EBPR.• Feast-famine regime enriches polyphosphate-accumulating organisms (PAOs).• Microfluidics replace sequencing batch reactors for aerobic granular sludge research.

Fully exploiting the potential of enzymes in cell-free biocatalysis requires stabilization of the catalytically active proteins and their integration into efficient reactor systems. Although in recent years initial steps towards the immobilization of such biomolecules in metal-organic frameworks (MOFs) have been taken, these demonstrations have been limited to batch experiments and to aqueous conditions. Here we demonstrate a MOF-based continuous flow enzyme reactor system, with high productivity and stability, which is also suitable for organic solvents. Under aqueous conditions, the stability of the enzyme was increased 30-fold, and the space-time yield exceeded that obtained with other enzyme immobilization strategies by an order of magnitude. Importantly, the infiltration of the proteins into the MOF did not require additional functionalization, thus allowing for time- and cost-efficient fabrication of the biocatalysts using label-free enzymes.

All-enzyme hydrogel (AEH) particles with a hydrodynamic diameter of up to 120 nm were produced intracellularly with an Escherichia coli-based in vivo system. The inCell-AEH nanoparticles were generated from polycistronic vectors enabling simultaneous expression of two interacting enzymes, the Lactobacillus brevis alcohol dehydrogenase (ADH) and the Bacillus subtilis glucose-1-dehydrogenase (GDH), fused with a SpyCatcher or SpyTag, respectively. Formation of inCell-AEH was analyzed by dynamic light scattering and atomic force microscopy. Using the stereoselective two-step reduction of a prochiral diketone substrate, we show that the inCell-AEH approach can be advantageously used in whole-cell flow biocatalysis, by which flow reactors could be operated for >4 days under constant substrate perfusion. More importantly, the inCell-AEH concept enables the recovery of efficient catalyst materials for stable flow bioreactors in a simple and economical one-step procedure from crude bacterial lysates. We believe that our method will contribute to further optimization of sustainable biocatalytic processes.

Natural evolution has produced an almost infinite variety of microorganisms that can colonize almost any conceivable habitat. Since the vast majority of these microbial consortia are still unknown, there is a great need to elucidate this “microbial dark matter” (MDM) to enable exploitation in biotechnology. We report the fabrication and application of a novel device that integrates a matrix of macroporous elastomeric silicone foam (MESIF) into an easily fabricated and scalable chip design that can be used for decoding MDM in environmental microbiomes. Technical validation, performed with the model organism Escherichia coli expressing a fluorescent protein, showed that this low-cost, bioinert, and widely modifiable chip is rapidly colonized by microorganisms. The biological potential of the chip was then illustrated through targeted sampling and enrichment of microbiomes in a variety of habitats ranging from wet, turbulent moving bed biofilters and wastewater treatment plants to dry air-based environments. Sequencing analyses consistently showed that MESIF chips are not only suitable for sampling with high robustness but also that the material can be used to detect a broad cross section of microorganisms present in the habitat in a short time span of a few days. For example, results from the biofilter habitat showed efficient enrichment of microorganisms belonging to the enigmatic Candidate Phyla Radiation, which comprise ∼70% of the MDM. From dry air, the MESIF chip was able to enrich a variety of members of Actinobacteriota, which is known to produce specific secondary metabolites. Targeted sampling from a wastewater treatment plant where the herbicide glyphosate was added to the chip’s reservoir resulted in enrichment of Cyanobacteria and Desulfobacteria, previously associated with glyphosate degradation. These initial case studies suggest that this chip is very well suited for the systematic study of MDM and opens opportunities for the cultivation of previously unculturable microorganisms.

We present an efficient and readily applicable strategy for the covalent ligation of proteins to DNA origami by using the SpyCatcher-SpyTag (SC-ST) connector system. This approach showed orthogonality with other covalent connectors and has been used exemplarily for the immobilization and study of stereoselective ketoreductases to gain insight into the spatial arrangement of enzymes on DNA nanostructures.

Continuous flow systems for chemical synthesis are becoming a major focus in organic chemistry and there is a growing interest in the integration of biocatalysts due to their high regio‐ and stereoselectivity. Methods established for 3D bioprinting enable the fast and simple production of agarose‐based modules for biocatalytic reactors if thermally stable enzymes are available. We report here on the characterization of four different cofactor‐free phenacrylate decarboxylase enzymes suitable for the production of 4‐vinylphenol and test their applicability for the encapsulation and direct 3D printing of disk‐shaped agarose‐based modules that can be used for compartmentalized flow microreactors. Using the most active and stable phenacrylate decarboxylase from Enterobacter spec. in a setup with four parallel reactors and a subsequent palladium(II) acetate‐catalysed Heck reaction, 4‐hydroxystilbene was synthesized from p‐coumaric acid with a total yield of 14.7 % on a milligram scale. We believe that, due to the convenient direct immobilization of any thermostable enzyme and straightforward tuning of the reaction sequence by stacking of modules with different catalytic activities, this simple process will facilitate the establishment and use of cascade reactions and will therefore be of great advantage for many research approaches., 3D printing in flow chemistry: 4‐Hydroxystilbene is obtained from p‐coumaric acid employing a phenacrylate decarboxylase enzyme immobilized in 3D‐printed agarose‐based flow reactor modules and a subsequent palladium(II) acetate‐catalyzed Heck reaction with a total yield of 14.7 % on a milligram scale.

Rapid tests for glucose-6-phosphate dehydrogenase (G6PD) are extremely important for determining G6PD deficiency, a widespread metabolic disorder which triggers hemolytic anemia in response to primaquine and tafenoquine medication, the most effective drugs for the radical cure of malaria caused by Plasmodium parasites. Current point-of-care diagnostic devices for G6PD are either qualitative, do not normalize G6PD activity to the hemoglobin concentration, or are very expensive. In this work we developed a capillary-driven microfluidic chip to perform a quantitative G6PD test and a hemoglobin measurement within 2 minutes and using less than 2 μL of sample. We used a powerful microfluidic module to integrate and resuspend locally the reagents needed for the G6PD assay and controls. We also developed a theoretical model that successfully predicts the enzymatic reactions on-chip, guides on-chip reagent spotting and allows efficient integration of multiple assays in miniaturized formats with only a few nanograms of reagents.

The development of a DNA-based cell-responsive biohybrid interface that can be used for spatially confined release of molecular cargo is reported. To this end, tailored DNA–protein conjugates are designed as gatekeepers that can be specifically cleaved by matrix metalloproteases (MMPs), which are secreted by many cancer cells. These gatekeepers can be installed by DNA hybridization on the surface of mesoporous silica nanoparticles (MSNs). The MSNs display another orthogonal DNA oligonucleotide that can be exploited for site-selective immobilization on solid glass surfaces to yield micropatterned substrates for cell adhesion. Using the human fibrosarcoma cell line HT1080 that secretes MMPs, it is demonstrated that the biohybrid surface is specifically modified by the cells to release both MSN-bound gatekeeper proteins and the encapsulated cargo peptide KLA. In view of the enormously high modularity of the system presented here, this approach promising for applications in drug delivery, tissue engineering, or other areas of nanobiotechnology is considered.

We here report the application of a machine-based microfluidic biofilm cultivation and analysis platform for studying the performance of biocatalytically active biofilms. By using robotic sampling, we succeeded in spatially resolving the productivity of three microfluidic reactors containing biocatalytically active biofilms that inducibly overexpress recombinant enzymes. Escherichia coli biofilms expressing two stereoselective oxidoreductases, the (R)-selective alcohol dehydrogenase LbADH and the (S)-selective ketoreductase Gre2p, as well as the phenolic acid decarboxylase EsPAD were used. The excellent reproducibility of the cultivation and analysis methods observed for all three systems underlines the usefulness of the new technical platform for the investigation of biofilms. In addition, we demonstrated that the analytical platform also opens up new opportunities to perform in-depth spatially resolved studies on the biomass growth in a reactor channel and its biochemical productivity. Since the platform not only offers the detailed biochemical characterization but also broad capabilities for the morphological study of living biofilms, we believe that our approach can also be performed on many other natural and artificial biofilms to systematically investigate a wide range of process parameters in a highly parallel manner using miniaturized model systems, thus advancing the harnessing of microbial communities for technical purposes.

Forming hydrogels with precise geometries is challenging and mostly done using photopolymerization, which involves toxic chemicals, rinsing steps, solvents, and bulky optical equipment. Here, we introduce a new method for in situ formation of hydrogels with a well‐defined geometry in a sealed microfluidic chip by interfacial polymerization. The geometry of the hydrogel is programmed by microfluidic design using capillary pinning structures and bringing into contact solutions containing hydrogel precursors from vicinal channels. The characteristics of the hydrogel (mesh size, molecular weight cut‐off) can be readily adjusted. This method is compatible with capillary‐driven microfluidics, fast, uses small volumes of reagents and samples, and does not require specific laboratory equipment. Our approach creates opportunities for filtration, hydrogel functionalization, and hydrogel‐based assays, as exemplified by a rapid, compact competitive immunoassay that does not require a rinsing step., PEG‐based hydrogels can be precisely localized in capillary‐driven microfluidic chips by in situ interfacial polymerization. This allows using hydrogels for filtration, studying diffusion processes of solutes across the hydrogels and rapid bioassays.

To apply enzymes in technical processes, a detailed understanding of the molecular mechanisms is required. Kinetic and thermodynamic parameters of enzyme catalysis are crucial to plan, model, and implement biocatalytic processes more efficiently. While the kinetic parameters, Km and kcat, are often accessible by optical methods, the determination of thermodynamic parameters requires more sophisticated methods. Isothermal titration calorimetry (ITC) allows the label-free and highly sensitive analysis of kinetic and thermodynamic parameters of individual steps in the catalytic cycle of an enzyme reaction. However, since ITC is susceptible to interferences due to denaturation or agglomeration of the enzymes, the homogeneity of the enzyme sample must always be considered, and this can be accomplished by means of dynamic light scattering (DLS) analysis. We here report on the use of an ITC-dependent work flow to determine both the kinetic and the thermodynamic data for a cofactor-dependent enzyme. Using a standardized approach with the implementation of sample quality control by DLS, we obtain high-quality data suitable for the advanced modeling of the enzyme reaction mechanism. Specifically, we investigated stereoselective reactions catalyzed by the NADPH-dependent ketoreductase Gre2p under different reaction conditions. The results revealed that this enzyme operates with an ordered sequential mechanism and is affected by substrate or product inhibition depending on the reaction buffer. Data reproducibility is ensured by specifying standard operating procedures, using programmed workflows for data analysis, and storing all data in a F.A.I.R. (findable, accessible, interoperable, and reusable) repository (https://doi.org/10.15490/fairdomhub.1.investigation.464.1). Our work highlights the utility for combined binding and kinetic studies for such complex multisubstrate reactions.

Protein micropatterning is a powerful tool for spatial arrangement of transmembrane and intracellular proteins in living cells. The restriction of one interaction partner (the bait, e.g., the receptor) in regular micropatterns within the plasma membrane and the monitoring of the lateral distribution of the bait&rsquo, s interaction partner (the prey, e.g., the cytosolic downstream molecule) enables the in-depth examination of protein-protein interactions in a live cell context. This study reports on potential pitfalls and difficulties in data interpretation based on the enrichment of clathrin, which is a protein essential for clathrin-mediated receptor endocytosis. Using a highly modular micropatterning approach based on large-area micro-contact printing and streptavidin-biotin-mediated surface functionalization, clathrin was found to form internalization hotspots within the patterned areas, which, potentially, leads to unspecific bait/prey protein co-recruitment. We discuss the consequences of clathrin-coated pit formation on the quantitative analysis of relevant protein-protein interactions, describe controls and strategies to prevent the misinterpretation of data, and show that the use of DNA-based linker systems can lead to the improvement of the technical platform.

A multiscale polydimethylsiloxane (PDMS) chip is presented, which provides an array of mesoscale open wells for cell culturing and, as unique feature, an array of microscale 1 mu m deep channels to fluidically connect neighboring wells. As demonstrated with SH-SY5Y cells, the small dimensions of the channels prevent migration of the cell soma but allow physical contacts established by the outgrowth of protoplasmic protrusions between cells in adjacent wells. Another important feature is the chip's mountability on solid substrates, such as glass. This enables the use of substrates previously patterned with biomolecules, as demonstrated by DNA-directed immobilization of proteins inside the reactor wells. Given the versatile addressability of cells, whether through surface-bound or inkjet-based administration of bioactive substances, it is believed that the reactor could be used for research in cell-cell communication networks, for example, in neurodegenerative diseases such as Alzheimer's disease.

We report on the directed postsynthetic functionalization of soft DNA nanocomposite materials with proteins. Using the example of the functionalization of silica nanoparticle‐modified DNA polymer materials with agonists or antagonists of the epidermal growth factor receptor EGFR cell membrane receptor, we demonstrate that hierarchically structured interfaces to living cells can be established. Owing to the modular design principle, even complex DNA nanostructures can be integrated into the materials, thereby enabling the high‐precision arrangement of ligands on the lower nanometer length scale. We believe that such complex biohybrid material systems can be used for new applications in biotechnology., Hierarchically structured interfaces to living cells can be established by functionalizing nanoparticle‐modified DNA polymer materials with agonists or antagonists of transmembrane receptors.

ChemPlusChem 85(3), 587 - 594 (2020). doi:10.1002/cplu.202000045, Published by Wiley, Malden Mass.

We present a facile method for the combined synthesis and purification of protein-decorated DNA origami nanostructures (DONs). DONs bearing reductively cleavable biotin groups in addition to ligands for ligation of recombinant proteins are bound to magnetic beads. Protein immobilization is conducted with a large protein excess to achieve high ligation yields. Subsequent to cleavage from the solid support, pure sample solutions are obtained which are suitable for direct AFM analysis of occupation patterns. We demonstrate the method's utility using three different orthogonal ligation methods, the "halo-based oligonucleotide binder" (HOB), a variant of Halo-tag, the "SpyTag/SpyCatcher" (ST/SC) system, and the enzymatic "ybbR tag" coupling. We find surprisingly low efficiency for ST/SC ligation, presumably due to electrostatic repulsion and steric hindrance, whereas the ybbR method, despite its ternary nature, shows good ligation yields. Our method is particularly useful for the development of novel ligation methods and the synthesis of mechanically fragile DONs that present protein patterns for surface-based cell assays.

All-enzyme hydrogels are efficient reagents for continuous flow biocatalysis. These materials can be obtained by self-assembly of two oligomeric enzymes, modified with the complementary SpyTag and SpyCatcher units. To facilitate access to the large proportion of biocatalytically relevant monomeric enzymes, we demonstrate that the tagging valency of the monomeric (S)-stereoselective ketoreductase Gre2p from Saccharomyces cerevisiae can be designed to assemble stable, active hydrogels with the cofactor-regenerating glucose 1-dehydrogenase GDH from Bacillus subtilis. Mounted in microfluidic reactors, these gels revealed high conversion rates and stereoselectivity in the reduction of prochiral methylketones under continuous flow for more than 8 days. The sequential use as well as parallelization by ‘numbering up’ of the flow reactor modules demonstrate that this approach is suitable for syntheses on the semipreparative scale.

Although DNA nanotechnology has developed into a highly innovative and lively field of research at the interface between chemistry, materials science, and biotechnology, there is still a great need for methodological approaches for bridging the size regime of DNA nanostructures with that of micrometer‐ and millimeter‐sized units for practical applications. We report on novel hierarchically structured composite materials from silica nanoparticles and DNA polymers that can be obtained by self‐assembly through the clamped hybridization chain reaction. The nanocomposite materials can be assembled into thin layers within microfluidically generated water‐in‐oil droplets to produce mechanically stabilized hollow spheres with uniform size distributions at high throughput rates. The fact that cells can be encapsulated in these microcontainers suggests that our concept not only contributes to the further development of supramolecular bottom‐up manufacturing, but can also be exploited for applications in the life sciences., Hierarchically structured composite materials made from silica nanoparticles and DNA polymers (red) can be self‐assembled by the clamped hybridization chain reaction into thin layers within microfluidically generated water‐in‐oil droplets to produce mechanically stabilized micrometer‐sized hollow spheres that hold potential for applications in the life sciences.

Biofilms are the natural form of life of the majority of microorganisms. These multispecies consortia are intensively studied not only for their effects on health and environment but also because they have an enormous potential as tools for biotechnological processes. Further exploration and exploitation of these complex systems will benefit from technical solutions that enable integrated, machine-assisted cultivation and analysis. We here introduce a microfluidic platform, where readily available microfluidic chips are connected by automated liquid handling with analysis instrumentation, such as fluorescence detection, microscopy, chromatography and optical coherence tomography. The system is operable under oxic and anoxic conditions, allowing for different gases and nutrients as feeding sources and it offers high spatiotemporal resolution in the analysis of metabolites and biofilm composition. We demonstrate the platform’s performance by monitoring the productivity of biofilms as well as the spatial organization of two bacterial species in a co-culture, which is driven by chemical gradients along the microfluidic channel. Helmholtz program BioInterfaces in Technology and Medicine

Cells from most mammalian tissues require an extracellular matrix (ECM) for attachment and proper functioning. In vitro cell cultures therefore must be supplied with an ECM that satisfies both the biological needs of cells used and the technical demands of the experimental setup. The latter include matrix functionalization for cell attachment, favorable microscopic properties, and affordable production costs. Here, modified DNA materials are therefore developed as an ECM mimic. The material is prepared by chemical cross-linking of commonly available salmon sperm DNA. To render the material cell-compatible, it is enzymatically modified by DNA polymerase I to provide versatile attachment points for peptides, proteins, or antibodies via a modular strategy. Different cells specifically attach to the material, even from mixed populations. They can be mildly released for further cell studies by DNase I-mediated digestion of the DNA material. Additionally, neural stem cells not only attach and survive on the material but also differentiate to a neural lineage when prompted. Furthermore, the DNA material can be employed to capture and retain cells under flow conditions. The simple preparation of the DNA material and its wide scope of applications open new perspectives for various cell study challenges and medical applications. published

The recommendations of the panel of experts on the further development of the ‘High-Tech Strategy’ of the Government of the Federal Republic of Germany designate the biologization of economic processes along with digitization as the central driver of societal change. Various fields such as the 'biologization of materials research' were then defined in the 'Bio-Agenda' in order to walk the path from biology to innovation. In view of this perspective, we describe how the use of biological materials and mechanisms can lead to resource conservation, the production of intelligent materials and new technological and medical applications. Our strategy, based on research on ‘Biointerfaces in Technology and Medicine’, aims at the development of modular biohybrid systems that could be used as 'biofactories of the future' for sustainable production processes. To achieve this goal, in a first phase already known technologies have to be further refined and integrated in order to obtain and apply compartmentalized reaction systems on different length and time scales. In a second phase, the resulting functional units will be employed to develop dissipative systems useful for biomedical applications and advanced production processes. From a technical point of view, future success in creating 'something entirely new' will depend crucially on robust and complementary research capabilities. Since the close connection of engineering and life sciences at KIT provides an excellent basis for this endeavor, we consider the above perspectives to be feasible.

[EN] The lithium ion recognition is receiving significant attention because of its application in pharmaceuticals, lubricants and, especially, in energy technology. We present a nanofluidic device for specific lithium ion recognition via host guest complexation in a confined environment. A lithium-selective receptor molecule, the aminoethyl-benzo-12-crown-4 (BC12C4-NH2), is designed and functionalized on single conical nanopores in polyethylene terephthalate (PET) membranes. The native carboxylic acid groups on the pore walls are covalently linked with the crown ether moieties and the process is monitored from the changes in the current voltage (I-V) curves. The B12-crown-4 moieties are known to specifically bind with lithium ions and when the modified pore is exposed to different alkali metal chloride solutions separately, significant changes in the ion current and rectification are only observed for lithium chloride. This fact suggests the generation of positively charged B12C4-Li+ complexes on the pore surface. Furthermore, the nanofluidic diode is able to recognize the lithium ion even in the presence of high concentrations of potassium ions in the external electrolyte solution. Thus, this nanodevice suggests a strategy to miniaturize nanofluidic porous systems for efficient recognition, extraction, and separation of lithium from raw materials., M.A., S.N., and W.E. acknowledge the funding from the Hessen State Ministry of Higher Education, Research and the Arts, Germany, under the LOEWE Project iNAPO. P.R and S.M. acknowledge financial support by the Spanish Ministry of Economic Affairs and Competitiveness (Grant MAT2015-65011-P) and FEDER. I.A. and C.M.N. acknowledge financial support through the Helmholtz Programme BioInterfaces in Technology and Medicine. The authors are thankful to Prof. Christina Trautmann from GSI (Department of Material Research) for support with the heavy ion irradiation experiments.

We describe the construction of binary self-assembling all-enzyme hydrogels that are comprised entirely of two tetrameric globular enzymes, the stereoselective dehydrogenase LbADH and the cofactor-regenerating glucose 1-dehydrogenase GDH. The enzymes were genetically fused with a SpyTag or SpyCatcher domain, respectively, to generate two complementary homo-tetrameric building blocks that polymerise under physiological conditions into porous hydrogels. The biocatalytic gels were used for the highly stereoselective reduction of a prochiral diketone substrate where they showed the typical behaviour of the coupled kinetics of coenzyme regenerating reactions in the substrate channelling regime. They effectively sequestrate the NADPH cofactor even under continuous flow conditions. Owing to their sticky nature, the gels can be readily mounted in simple microfluidic reactors without the need for supportive membranes. The reactors revealed extraordinary high space-time yields with nearly quantitative conversion (>95%), excellent stereoselectivity (d.r. > 99:1), and total turnover numbers of the expensive cofactor NADP(H) that are amongst the highest values ever reported.

A generic methodology for cytosolic delivery of large supramolecular multiprotein complexes into living cells is described that takes advantage of the highly-controllable bottom-up fabrication of protein-decorated DNA nanostructures and the microfluidic “cell squeezing” technique. Therein, cells are deformed upon passage through a narrow constriction leading to formation of transient holes in the cell membrane that enable the diffusion of the protein-DNA nanostructures from the surrounding buffer into the cytosol. A diverse set of multiprotein complexes was assembled on DNA origami nanostructures using streptavidin and the sensitive glucose sensor protein FLIP as model systems. We demonstrate that our approach allows for the direct cytosolic delivery of these multifunctional protein complexes into the cytosol of HeLa cells. We also demonstrate that targeting groups can be incorporated into the protein-DNA nanoassemblies to enable their intracellular targeting to cytosolic compartments, such as the cytoskeleton or nucleus. We believe that this methodology will open up novel strategies for research in fundamental cell biology, such as the reverseengineering of the supramolecular machinery involved in gene regulation, cell signalling, or cell division. Furthermore, direct applications in immunotherapy can be foreseen.

Biological compartmentalization is a fundamental principle of life that allows cells to metabolize, propagate, or communicate with their environment. Much research is devoted to understanding this basic principle and to harness biomimetic compartments and catalytic cascades as tools for technological processes. This Review summarizes the current state-of-the-art of these developments, with a special emphasis on length scales, mass transport phenomena, and molecular scaffolding approaches, ranging from small cross-linkers over proteins and nucleic acids to colloids and patterned surfaces. We conclude that the future exploration and exploitation of these complex systems will largely benefit from technical solutions for the integrated, machine-assisted development and maintenance of a next generation of biotechnological processes. These goals should be achievable by implementing microfluidics, robotics, and added manufacturing techniques supplemented by theoretical simulations as well as computer-aided process modeling based on big data obtained from multiscale experimental analyses.

[EN] We demonstrate experimentally and theoretically a nanofluidic device for the selective recognition of the cesium ion by exploiting host¿guest interactions inside confined geometry. For this purpose, a host molecule, i.e., the amine-terminated p-tert-butylcalix[4]arene-crown (tBuC[4]C¿NH2), is successfully synthesized and functionalized on the surface of a single conical nanopore fabricated in a poly(ethylene terephthalate) (PET) membrane through carbodiimide coupling chemistry. On exposure to the cesium cation, the t-BuC[4]C¿Cs+ complex is formed through host¿guest interaction, leading to the generation of positive fixed charges on the pore surface. The asymmetrical distribution of these groups along the conical nanopore leads to the electrical rectification observed in the current¿voltage (I¿V) curve. On the contrary, other alkali cations are not able to induce any significant change in the rectification characteristics of the nanopore. The success of the chemical modification is monitored from the changes in the electrical readout of the nanopore. Theoretical results based on the Nernst¿Planck and Poisson equations further demonstrate the validity of the experimental approach to the cesium-induced ionic conduction of the nanopore., M.A., S.N., and W.E. acknowledge funding from the Hessen State Ministry of Higher Education, Research and the Arts, Germany, under LOEWE project iNAPO. P.R., J.C., and S.M. acknowledge financial support by the Spanish Ministry of Economic Affairs and Competitiveness (MAT2015-65011-P) and FEDER. I.A. and C.M.N. acknowledge financial support through the Helmholtz programme Biolnterfaces in Technology and Medicine. The authors are thankful to Prof. Christina Trautmann from GSI (Department of Material Research) for support with the heavy ion irradiation experiments and Dr. M. Nawaz Tahir (Johannes Gutenberg University Mainz) for help with contact angle measurements.

We herein describe the engineering of E. coli strains that display orthogonal tags for immobilization on their surface and overexpress a functional heterologous “protein content” in their cytosol at the same time. Using the outer membrane protein Lpp-ompA, cell-surface display of the streptavidin-binding peptide, the SpyTag/SpyCatcher system, or a HaloTag variant allowed us to generate bacterial strains that can selectively bind to solid substrates, as demonstrated with magnetic microbeads. The simultaneous cytosolic expression of functional content was demonstrated for fluorescent proteins or stereoselective ketoreductase enzymes. The latter strains gave high selectivities for specific immobilization onto complementary surfaces and also in the whole-cell stereospecific transformation of a prochiral CS-symmetric nitrodiketone.

Progress in the development of protein‐immobilization strategies and methods has made protein biochips increasingly accessible. The integration of these assay and analysis platforms into biomedical and biotechnological research has substantially expanded the repertoire of methods available for proteomics and biomarker research and for drug development. This Minireview highlights selected developments in the application of protein biochips in these fields., Biochip whys and wherefores: Protein biochips are becoming increasingly accessible. But what are they currently being used for? The integration of these assay and analysis platforms into biomedical and biotechnological research has substantially expanded the repertoire of methods available for proteomics and biomarker research and drug development.WILEY-VCH

Today, DNA nanotechnology is one of the methods of choice to achieve spatiotemporal control of matter at the nanoscale. By combining the peculiar spatial addressability of DNA origami structures with the switchable mechanical movement of small DNA motifs, we constructed reconfigurable DNA nanochambers as dynamic compartmentalization systems. The reversible extension and contraction of the inner cavity of the structures was used to control the distance-dependent energy transfer between two preloaded fluorophores. Interestingly, single-molecule FRET studies revealed that the kinetics of the process are strongly affected by the choice of the switchable motifs and/or actuator sequences, thus offering a valid method for fine-tuning the dynamic properties of large DNA nanostructures. We envisage that the proposed DNA nanochambers may function as model structures for artificial biomimetic compartments and transport systems.

Cell phenotype is determined by protein network states that are maintained by the dynamics of multiple protein interactions.1 Fluorescence microscopy approaches that measure protein interactions in individual cells, such as by Forster resonant energy transfer (FRET), are limited by the spectral separation of fluorophores and thus are most suitable to analyze a single protein interaction in a given cell. However, analysis of correlations between multiple protein interactions is required to uncover the interdependence of protein reactions in dynamic signal networks. Available protein-array technologies enable the parallel analysis of interacting proteins from cell extracts, however, they can only provide a single snapshot of dynamic interaction networks. Moreover, because of the high level of variance from cell to cell in protein expression levels and reaction state, cell extracts only provide an average measure of protein interaction states and therefore the detection of the relations between proteins is blurred. As an intermediate step, a visual immunoprecipitation assay was developed that allowed direct observation of multiple, dynamic protein interactions on immobilized, distinguishable beads in cell extracts.2 A microstructuring approach allowed for analysis of the interaction of one naturally occurring receptor type with one of its interaction partners inside cells.3 To analyze multiple protein interactions inside a single living cell, multiple receptors must be arranged in a defined pattern to distinguish their identity. Herein, we developed a general strategy to generate protein arrays with multiple arbitrary bait proteins by way of artificial-receptor constructs at sub-cellular feature size and applied this technology to simultaneously measure two-protein interaction kinetics inside an individual living cell.

Protective proteases are key elements of protein quality control pathways that are up-regulated, for example, under various protein folding stresses. These proteases are employed to prevent the accumulation and aggregation of misfolded proteins that can impose severe damage to cells. The high temperature requirement A (HtrA) family of serine proteases has evolved to perform important aspects of ATP-independent protein quality control. So far, however, no HtrA protease is known that degrades protein aggregates. We show here that human HTRA1 degrades aggregated and fibrillar tau, a protein that is critically involved in various neurological disorders. Neuronal cells and patient brains accumulate less tau, neurofibrillary tangles, and neuritic plaques, respectively, when HTRA1 is expressed at elevated levels. Furthermore, HTRA1 mRNA and HTRA1 activity are up-regulated in response to elevated tau concentrations. These data suggest that HTRA1 is performing regulated proteolysis during protein quality control, the implications of which are discussed. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.

Finding the optimal combinations of ligands for tissue-specific delivery is tedious even if only a few well-established compounds are tested. The cargo affects the receptor-ligand interaction, especially when it is charged like DNA. The ligand should therefore be evaluated together with its cargo. Several viruses have been shown to interact with more than one receptor, for efficient internalization. We here present a DNA oligonucleotide-based method for inexpensive and rapid screening of biotin labeled ligands for combinatorial effects on cellular binding and uptake. The oligonucleotide complex was designed as a 44 bp double-stranded DNA oligonucleotide with one central streptavidin molecule and a second streptavidin at the terminus. The use of a highly advanced robotic platform ensured stringent processing and execution of the experiments. The oligonucleotides were fluorescently labeled and used for detection and analysis of cell-bound, internalized and intra-cellular compartmentalized constructs by an automated line-scanning confocal microscope, IN Cell Analyzer 3000. All possible combinations of 22 ligands were explored in sets of 2 and tested on 6 different human cell lines in triplicates. In total, 10 000 transfections were performed on the automation platform. Cell-specific combinations of ligands were identified and their relative position on the scaffold oligonucleotide was found to be of importance. The ligands were found to be cargo dependent, carbohydrates were more potent for DNA delivery whereas cell penetrating peptides were more potent for delivery of less charged particles. © 2008 American Institute of Physics.

An assay for very sensitive antigen detection is described which takes advantage of the self- assembly capabilities of semi-synthetic conjugates of DNA and proteins. The general scheme of this assay is similar to a two-sided (sandwich) enzyme-linked immunoassay (ELISA); however, covalent single-stranded DNA-streptavidin (STV) conjugates, capable of hybridizing to complementary surface-bound DNA oligomers, are utilized for the effective immobilization of either capture antibodies or antigens, rather than the chemi- or physisorption usually applied in ELISA. Immuno-PCR (IPCR) is employed as a method for signal generation, utilizing oligomeric reagents obtained by self-assembly of STV, biotinylated DNA and antibodies. In three different model systems, detecting human IgG, rabbit IgG or carcinoembryonic antigen, this combination allowed one to increase the sensitivity of the analogous ELISA approximately 1000-fold. For example

The self-assembly of bis-biotinylated double-stranded DNA and the tetravalent biotin-binding protein streptavidin (STV) have been studied by non-denaturing gel electrophoresis and atomic force microscopy (AFM). The rapid self-assembly reproducibly generated populations of individual oligomeric complexes. Most strikingly, the oligomers predominantly contained bivalent STV molecules bridging two adjacent DNA fragments to form linear nanostructures. Trivalent STV branch points occurred with a lower frequency and the presence of tetravalent STV was scarce. However, valency distribution, size and the exchange dynamics of the supramolecular aggregates were highly sensitive to stoichiometric variations in the relative molar coupling ratio of bis-biotinylated DNA and STV. The largest aggregates were obtained from equimolar amounts while excess STV led to the formation of smaller oligomers appearing as fingerprint-like band patterns in electrophoresis. Excess DNA, however, induces a complete breakdown of the oligomers, likely a consequence of the instability of STV conjugates containing more than two biotinylated DNA fragments. It was demonstrated that the oligomers can further be functionalized, for instance by the coupling of biotinylated immunoglobulins. Both pure and also antibody-modified DNA-STV oligomers were used as reagents in immuno-PCR (IPCR), a highly sensitive detection method for proteins and other antigens. Employment of the supramolecular reagents led to an approximately 100-fold enhanced sensitivity compared to the conventional IPCR procedure.

Modified biomolecules were used for the non-covalent assembly of novel bioconjugates. Hybrid molecules were synthesized from short single-stranded DNA and streptavidin by chemical methods using a heterobispecific crosslinker. The covalent attachment of an oligonucleotide moiety to streptavidin provides a specific recognition domain for a complementary nucleic acid sequence, in addition to the four native biotin-binding sites. These bispecific binding capabilities allow the hybrid molecules to serve as versatile connectors in a variety of applications. Bifunctional constructs have been prepared from two complementary hybrid molecules, each previously conjugated to biotinylated immunoglobulin G or alkaline phosphatase. The use of nucleic acid sequences as a template for the formation of an array of proteins is further demonstrated on two size scales. A macroscopic DNA array on a microtiter plate has been transformed into a comparable protein chip. A nano-scale array was made by hybridizing DNA-tagged proteins to specific positions along a RNA or DNA sequence. The generation of supramolecular bioconjugates was shown by quantitative measurements and gel-retardation assays.