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The nanoscale arrangement of ligands can have a major effect on the activation of membrane receptor proteins and thus cellular communication mechanisms. Here we report on the technological development and use of tailored DNA origami-based molecular rulers to fabricate "Multiscale Origami Structures As Interface for Cells" (MOSAIC), to enable the systematic investigation of the effect of the nanoscale spacing of epidermal growth factor (EGF) ligands on the activation of the EGF receptor (EGFR). MOSAIC-based analyses revealed that EGF distances of about 30-40 nm led to the highest response in EGFR activation of adherent MCF7 and Hela cells. Our study emphasizes the significance of DNA-based platforms for the detailed investigation of the molecular mechanisms of cellular signaling cascades.
Assuntos
Fator de Crescimento Epidérmico , Receptores ErbB , Humanos , DNA/química , Fator de Crescimento Epidérmico/química , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Células HeLa , Ligantes , Transdução de SinaisRESUMO
Artificial reconstruction of naturally evolved principles, such as compartmentalization and cascading of multienzyme complexes, offers enormous potential for the development of biocatalytic materials and processes. Due to their unique addressability at the nanoscale, DNA origami nanostructures (DON) have proven to be an exceptionally powerful tool for studying the fundamental processes in biocatalytic cascades. To systematically investigate the diffusion-reaction network of (co)substrate transfer in enzyme cascades, a model system of stereoselective ketoreductase (KRED) with cofactor regenerating enzyme is assembled in different spatial arrangements on DNA nanostructures and is located in the sphere of microbeads (MB) as a spatially confining nano- and microenvironment, respectively. The results, obtained through the use of highly sensitive analytical methods, Western blot-based quantification of the enzymes, and mass spectrometric (MS) product detection, along with theoretical modeling, provide strong evidence for the presence of two interacting compartments, the diffusion layers around the microbead and the DNA scaffold, which influence the catalytic efficiency of the cascade. It is shown that the microscale compartment exerts a strong influence on the productivity of the cascade, whereas the nanoscale arrangement of enzymes has no influence but can be modulated by the insertion of a diffusion barrier.
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DNA , Nanoestruturas , DNA/química , Nanoestruturas/química , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Biocatálise , CatáliseRESUMO
Chiral materials are essential to perceive photonic devices that control the helicity of light. However, the chirality of natural materials is rather weak, and relatively thick films are needed for noticeable effects. To overcome this limitation, artificial photonic materials were suggested to affect the chiral response in a much more substantial manner. Ideally, a single layer of such a material, a metasurface, should already be sufficient. While various structures fabricated with top-down nanofabrication technologies have already been reported, here we propose to utilize scaffolded DNA origami technology, a scalable bottom-up approach for metamolecule production, to fabricate a chiral metasurface. We introduce a chiral plasmonic metamolecule in the shape of a tripod and simulate its optical properties. By fixing the metamolecule to a rectangular planar origami, the tripods can be assembled into a 2D DNA origami crystal that forms a chiral metasurface. We simulate the optical properties but also fabricate selected devices to assess the experimental feasibility of the suggested approach critically.
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DNA , DNA/química , Ressonância de Plasmônio de Superfície/instrumentação , Nanotecnologia , Nanoestruturas/químicaRESUMO
DNA hydrogels hold significant promise for biomedical applications and can be synthesized through enzymatic Rolling Circle Amplification (RCA). Due to the exploratory nature of this emerging field, standardized RCA protocols specifying the impact of reaction parameters are currently lacking. This study varied template sequences and reagent concentrations, evaluating RCA synthesis efficiency and hydrogel mechanical properties through quantitative PCR (qPCR) and indentation measurements, respectively. Primer concentration and stabilizing additives showed minimal impact on RCA efficiency, while changes in polymerase and nucleotide concentrations had a stronger effect. Concentration of the circular template exerted the greatest influence on RCA productivity. An exponential correlation between hydrogel viscosity and DNA amplicon concentration was observed, with nucleobase sequence significantly affecting both amplification efficiency and material properties, particularly through secondary structures. This study suggests that combining high-throughput experimental methods with structural folding prediction offers a viable approach for systematically establishing structure-property relationships, aiding the rational design of DNA hydrogel material systems.
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Microorganisms serve as linchpins in agricultural systems. Classic examples include microbial composting for nutrient recovery, using microorganisms in biogas technology for agricultural waste utilization, and employing biofilters to reduce emissions from stables or improve water quality in aquaculture. This mini-review highlights the importance of microbiome analysis in understanding microbial diversity, dynamics, and functions, fostering innovations for a more sustainable agriculture. In this regard, customized microorganisms for soil improvement, replacements for harmful agrochemicals or antibiotics in animal husbandry, and (probiotic) additives in animal nutrition are already in or even beyond the testing phase for a large-scale conventional agriculture. Additionally, as climate change reduces arable land, new strategies based on closed-loop systems and controlled environment agriculture, emphasizing microbial techniques, are being developed for regional food production. These strategies aim to secure the future food supply and pave the way for a sustainable, resilient, and circular agricultural economy. KEY POINTS: ⢠Microbial strategies facilitate the integration of multiple trophic levels, essential for cycling carbon, nitrogen, phosphorus, and micronutrients. ⢠Exploring microorganisms in integrated biological systems is essential for developing practical agricultural solutions. ⢠Technological progress makes sustainable closed-entity re-circulation systems possible, securing resilient future food production.
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Agricultura , Microbiota , Agricultura/métodos , Agricultura/economia , Microbiologia do Solo , Animais , Desenvolvimento Sustentável , Criação de Animais Domésticos/métodos , Criação de Animais Domésticos/economia , Abastecimento de Alimentos , Bactérias/metabolismo , Bactérias/classificaçãoRESUMO
The natural micro- and nanoscale organization of biomacromolecules is a remarkable principle within living cells, allowing for the control of cellular functions by compartmentalization, dimensional diffusion and substrate channeling. In order to explore these biological mechanisms and harness their potential for applications such as sensing and catalysis, molecular scaffolding has emerged as a promising approach. In the case of synthetic enzyme cascades, developments in DNA nanotechnology have produced particularly powerful scaffolds whose addressability can be programmed with nanometer precision. In this minireview, we summarize recent developments in the field of biomimetic multicatalytic cascade reactions organized on DNA nanostructures. We emphasize the impact of the underlying design principles like DNA origami, efficient strategies for enzyme immobilization, as well as the importance of experimental design parameters and theoretical modeling. We show how DNA nanostructures have enabled a better understanding of diffusion and compartmentalization effects at the nanometer length scale, and discuss the challenges and future potential for commercial applications.
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Nanoestruturas , Ácidos Nucleicos , Nanotecnologia/métodos , Nanoestruturas/química , DNA/química , Enzimas Imobilizadas , Conformação de Ácido NucleicoRESUMO
The adhesion of circulating tumor cells (CTCs) to the endothelial lumen and their extravasation to surrounding tissues are crucial in the seeding of metastases and remain the most complex events of the metastatic cascade to study. Integrins expressed on CTCs are major regulators of the extravasation process. This knowledge is primarily derived from animal models and biomimetic systems based on artificial endothelial layers, but these methods have ethical or technical limitations. We present a versatile microfluidic device to study cancer cell extravasation that mimics the endothelial barrier by using a porous membrane functionalized with DNA origami nanostructures (DONs) that display nanoscale patterns of adhesion peptides to circulating cancer cells. The device simulates physiological flow conditions and allows direct visualization of cell transmigration through microchannel pores using 3D confocal imaging. Using this system, we studied integrin-specific adhesion in the absence of other adhesive events. Specifically, we show that the transmigration ability of the metastatic cancer cell line MDA-MB-231 is influenced by the type, distance, and density of adhesion peptides present on the DONs. Furthermore, studies with mixed ligand systems indicate that integrins binding to RGD (arginine-glycine-aspartic acid) and IDS (isoleucine-aspartic acid-serine) did not synergistically enhance the extravasation process of MDA-MB-231 cells.
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DNA , Células Neoplásicas Circulantes , Humanos , DNA/química , DNA/metabolismo , Células Neoplásicas Circulantes/patologia , Células Neoplásicas Circulantes/metabolismo , Linhagem Celular Tumoral , Técnicas Analíticas Microfluídicas , Nanoestruturas/química , Adesão Celular , Comunicação CelularRESUMO
Sustainable approaches to circular economy in animal agriculture are still poorly developed. Here, we report an approach to reduce gaseous emissions of CO2 and NH3 from animal housing while simultaneously using them to produce value-added biomass. To this end, a cone-shaped, helical photobioreactor was developed that can be integrated into animal housing by being freely suspended, thereby combining a small footprint with a physically robust design. The photobioreactor was coupled with the exhaust air of a chicken house to allow continuous cultivation of a mixed culture of Arthrospira spec. (Spirulina). Continuous quantification of CO2 and NH3 concentration showed that the coupled algae reactor effectively purifies the exhaust air from the chicken house while producing algal biomass. Typical production rates of greater than 0.3 g/l*day dry mass were obtained, and continuous operation was possible for several weeks. Morphological, biochemical, and genomic characterization of Spirulina cultures yielded insights into the dynamics and metabolic processes of the microbial community. We anticipate that further optimization of this approach will provide new opportunities for the generation of value-added products from gaseous CO2 and NH3 waste emissions, linking resource-efficient production of microalgae with simultaneous sequestration of animal emissions. KEY POINTS: ⢠Coupling a bioreactor with exhaust gases of chicken coop for production of biomass. ⢠Spirulina mixed culture removes CO2 and NH3 from chicken house emissions. ⢠High growth rates and biodiversity adaptation for nitrogen metabolism. Towards a sustainable circular economy in livestock farming. The functional coupling of a helical tube photobioreactor with exhaust air from a chicken house enabled the efficient cultivation of Spirulina microalgae while simultaneously sequestering the animals' CO2 and NH3 emissions.
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Microalgas , Spirulina , Animais , Gases/metabolismo , Dióxido de Carbono/metabolismo , Fotobiorreatores , Biomassa , Abrigo para Animais , Galinhas , Microalgas/metabolismoRESUMO
News from an old acquaintance: The streptavidin (STV)-biotin binding system is frequently used for the decoration of DNA origami nanostructures (DON) to study biological systems. Here, a surprisingly high dynamic of the STV/DON interaction is reported, which is affected by the structure of the DNA linker system. Analysis of different mono- or bi-dentate linker architectures on DON with a novel high-speed atomic force microscope (HS-AFM) enabling acquisition times as short as 50 ms per frame gave detailed insights into the dynamics of the DON/STV interaction, revealing dwell times in the sub-100 millisecond range. The linker systems are also used to present biotinylated epidermal growth factor on DON to study the activation of the epidermal growth factor receptor signaling cascade in HeLa cells. The studies confirm that cellular activation correlated with the binding properties of linker-specific STV/DON interactions observed by HS-AFM. This work sheds more light on the commonly used STV/DON system and will help to further standardize the use of DNA nanostructures for the study of biological processes.
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DNA , Nanoestruturas , DNA/química , Células HeLa , Humanos , Ligantes , Microscopia de Força Atômica , Nanoestruturas/química , Estreptavidina/químicaRESUMO
Enantiopure α-hydroxy ketones are important building blocks of active pharmaceutical ingredients (APIs), which can be produced by thiamine-diphosphate-dependent lyases, such as benzaldehyde lyase. Here we report the discovery of a novel thermostable benzaldehyde lyase from Rhodococcus erythropolis R138 (ReBAL). While the overall sequence identity to the only experimentally confirmed benzaldehyde lyase from Pseudomonas fluorescens Biovar I (PfBAL) was only 65 %, comparison of a structural model of ReBAL with the crystal structure of PfBAL revealed only four divergent amino acids in the substrate binding cavity. Based on rational design, we generated two ReBAL variants, which were characterized along with the wild-type enzyme in terms of their substrate spectrum, thermostability and biocatalytic performance in the presence of different co-solvents. We found that the new enzyme variants have a significantly higher thermostability (up to 22 °C increase in T50 ) and a different co-solvent-dependent activity. Using the most stable variant immobilized in packed-bed reactors via the SpyCatcher/SpyTag system, (R)-benzoin was synthesized from benzaldehyde over a period of seven days with a stable space-time-yield of 9.3â mmol â L-1 â d-1 . Our work expands the important class of benzaldehyde lyases and therefore contributes to the development of continuous biocatalytic processes for the production of α-hydroxy ketones and APIs.
Assuntos
Cetonas , Rhodococcus , Aldeído Liases/metabolismo , BenzaldeídosRESUMO
All-enzyme hydrogel (AEH) particles with a hydrodynamic diameter of up to 120â nm were produced intracellularly with an Escherichia coli-based inâ 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 >4â 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.
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Álcool Desidrogenase , Nanopartículas , Biocatálise , Álcool Desidrogenase/metabolismo , Escherichia coli/metabolismo , Reatores Biológicos , Enzimas Imobilizadas/metabolismoRESUMO
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.
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Microfluídica , Esgotos , Biofilmes , Reatores Biológicos , Hibridização in Situ Fluorescente , Fósforo , Polifosfatos , Eliminação de Resíduos LíquidosRESUMO
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.
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Enzimas Imobilizadas , Estruturas Metalorgânicas , Biocatálise , Catálise , Enzimas/metabolismo , Enzimas Imobilizadas/metabolismo , Estruturas Metalorgânicas/metabolismo , Proteínas/metabolismo , SolventesRESUMO
Combining structural DNA nanotechnology with the virtually unlimited variety of enzymes offers unique opportunities for generating novel biocatalytic devices. However, the immobilization of enzymes is still restricted by a lack of efficient covalent coupling techniques. The rational re-engineering of the genetically fusible SNAP-tag linker is reported here. By replacing five amino acids that alter the electrostatic properties of the SNAP_R5 variant, up to 11-fold increased coupling efficiency with benzylguanine-modified oligonucleotides and DNA origami nanostructures (DON) was achieved, resulting in typical occupancy densities of 75%. The novel SNAP_R5 linker can be combined with the equally efficient Halo-based oligonucleotide binding tag (HOB). Since both linkers exhibit neither cross-reactivity nor non-specific binding, they allowed orthogonal assembly of an enzyme cascade consisting of the stereoselective ketoreductase Gre2p and the cofactor-regenerating isocitrate dehydrogenase on DON. The cascade showed approximately 1.6-fold higher activity in a stereoselective cascade reaction than the corresponding free solubilized enzymes. The connector system presented here and the methods used to validate it represent important tools for further development of DON-based multienzyme systems to investigate mechanistic effects of substrate channeling and compartmentalization relevant for exploitation in biosensing and catalysis.
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Nanoestruturas , Biocatálise , DNA/metabolismo , Nanotecnologia , OligonucleotídeosRESUMO
Microfluidic devices can mimic naturally occurring microenvironments and create microbial population heterogeneities ranging from planktonic cells to biofilm states. The exposure of such populations to spatially organized stress gradients can promote their adaptation into complex phenotypes, which are otherwise difficult to achieve with conventional experimental setups. Here a microfluidic chip that employs precise chemical gradients in consecutive microcompartments to perform microbial adaptive laboratory evolution (ALE), a key tool to study evolution in fundamental and applied contexts is described. In the chip developed here, microbial cells can be exposed to a defined profile of stressors such as antibiotics. By modulating this profile, stress adaptation in the chip through resistance or persistence can be specifically controlled. Importantly, chip-based ALE leads to the discovery of previously unknown mutations in Escherichia coli that confer resistance to nalidixic acid. The microfluidic device presented here can enhance the occurrence of mutations employing defined micro-environmental conditions to generate data to better understand the parameters that influence the mechanisms of antibiotic resistance.
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Dispositivos Lab-On-A-Chip , Microfluídica , Antibacterianos/farmacologia , Biofilmes , Resistência Microbiana a Medicamentos/genética , Escherichia coli/genética , MutaçãoRESUMO
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.
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Biofilmes/crescimento & desenvolvimento , Escherichia coli/fisiologia , Dispositivos Lab-On-A-Chip , Técnicas Analíticas MicrofluídicasRESUMO
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.
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Nucleic acid hybridization reactions play an important role in many (bio)chemical fields, for example, for the development of portable point-of-care diagnostics, and often such applications require nucleic acid-based reaction systems that ideally run without enzymes under isothermal conditions. The use of novel capillary-driven microfluidic chips to perform two isothermal nucleic acid hybridization reactions, the simple opening of molecular beacon structures and the complex reaction cascade of a clamped-hybridization chain reaction (C-HCR), is reported here. For this purpose, reagents are arranged in a self-coalescence module (SCM) of a passive silicon microfluidic chip using inkjet spotting. The SCM occupies a footprint of ≈7 mm2 of a ≈0.4 × 2 cm2 microfluidic chip. By means of fluorophore-labeled DNA probes, the hybridization reactions can be analyzed in just ≈2 min and using only ≈3 µL of the sample. Furthermore, the SCM chip offers a variety of reagent delivery options, allowing, for example, the influence of the initiator concentration on the kinetics of C-HCR to be investigated systematically with minimal sample and time requirements. These results suggest that self-powered microfluidic chips equipped with a SCM provide a powerful platform for performing and investigating complex reaction systems.
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Técnicas Analíticas Microfluídicas , Ácidos Nucleicos , Corantes Fluorescentes , Microfluídica , Técnicas de Amplificação de Ácido Nucleico , Hibridização de Ácido NucleicoRESUMO
The display of complex proteins on the surface of cells is of great importance for protein engineering and other fields of biotechnology. Herein, we describe a modular approach, in which the membrane anchor protein Lpp-OmpA and a protein of interest (passenger) are expressed independently as genetically fused SpyCatcher and SpyTag units and assembled inâ situ by post-translational coupling. Using fluorescent proteins, we first demonstrate that this strategy allows the construct to be installed on the surface of E. coli cells. The scope of our approach was then demonstrated by using three different functional enzymes, the stereoselective ketoreductase Gre2p, the homotetrameric glucose 1-dehydrogenase GDH, and the bulky heme- and diflavin-containing cytochrome P450 BM3 (BM3). In all cases, the SpyCatcher-SpyTag method enabled the generation of functional whole-cell biocatalysts, even for the bulky BM3, which could not be displayed by conventional fusion with Lpp-OmpA. Furthermore, by using a GDH variant carrying an internal SpyTag, the system could be used to display an enzyme with unmodified N- and C-termini.
Assuntos
Técnicas de Visualização da Superfície Celular/métodos , Enzimas/metabolismo , Membrana Celular/metabolismo , Enzimas/genética , Escherichia coli/citologia , Escherichia coli/genética , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Dispersed negatively charged silica nanoparticles segregate inside microfluidic water-in-oil (W/O) droplets that are coated with a positively charged lipid shell. We report a methodology for the quantitative analysis of this self-assembly process. By using real-time fluorescence microscopy and automated analysis of the recorded images, kinetic data are obtained that characterize the electrostatically-driven self-assembly. We demonstrate that the segregation rates can be controlled by the installment of functional moieties on the nanoparticle's surface, such as nucleic acid and protein molecules. We anticipate that our method enables the quantitative and systematic investigation of the segregation of (bio)functionalized nanoparticles in microfluidic droplets. This could lead to complex supramolecular architectures on the inner surface of micrometer-sized hollow spheres, which might be used, for example, as cell containers for applications in the life sciences.