Identifying structure formation in semicrystalline conjugated polymers is the fundamental basis to understand electronic processes in these materials. Although correlations between physical ...properties, structure formation, and device parameters of regioregular, semicrystalline poly(3-hexylthiophene) (P3HT) have been established, it has remained difficult to disentangle the influence of regioregularity, polydispersity, and molecular weight. Here we show that the most commonly used synthetic protocol for the synthesis of P3HT, the living Kumada catalyst transfer polycondensation (KCTP) with Ni(dppp)Cl2 as the catalyst, leads to regioregular chains with one single tail-to-tail (TT) defect distributed over the whole chain, in contrast to the hitherto assumed exclusive location at the chain end. NMR end-group analysis and simulations are used to quantify this effect. A series of entirely defect-free P3HT materials with different molecular weights is synthesized via new, soluble nickel initiators. Data on structure formation in defect-free P3HT, as elucidated by various calorimetric and scattering experiments, allow the development of a simple model for estimating the degree of crystallinity. We find very good agreement for predicted and experimentally determined degrees of crystallinities as high as ∼70%. For Ni(dppp)Cl2-initiated chains comprising one distributed TT unit, the comparison of simulated crystallinities with calorimetric and optical measurements strongly suggests incorporation of the TT unit into the crystal lattice, which is accompanied by an increase in backbone torsion. Polydispersity is identified as a major parameter determining crystallinity within the molecular weight range investigated. We believe that the presented approach and results not only contribute to understanding structure formation in P3HT but are generally applicable to other semicrystalline conjugated polymers as well.
Spatiotemporal organization of distinct biological processes in cytomimetic compartments is a crucial step towards engineering functional artificial cells. Mimicking controlled bi‐directional ...molecular communication inside artificial cells remains a considerable challenge. Here we present photoswitchable molecular transport between programmable membraneless organelle‐like DNA coacervates in a synthetic microcompartment. We use droplet microfluidics to fabricate membraneless non‐fusing DNA coacervates by liquid‐liquid phase separation in a water‐in‐oil droplet, and employ the interior DNA coacervates as artificial organelles to imitate intracellular communication via photo‐regulated uni‐ and bi‐directional transfer of biomolecules. Our results highlight a promising new route to assembly of multicompartment artificial cells with functional networks.
Photoswitchable bi‐directional trafficking of biomolecules between programmable DNA‐based artificial membraneless organelles in a cell‐like microcompartment has been achieved to imitate intracellular communication towards construction of advanced functional artificial cells.
In this paper, we demonstrate a double nanoimprinting process that allows the formation of nanostructured polymer heterojunctions of composition and morphology that can be selected independently. We ...fabricated photovoltaic (PV) devices with extremely high densities (1014/mm2) of interpenetrating nanoscale columnar features in the active polymer blend layer. The smallest feature sizes are as small as 25 nm on a 50 nm pitch, which results in a spacing of heterojunctions at or below the exciton diffusion length. Photovoltaic devices based on double-imprinted poly((9,9-dioctylfluorene)-2,7-diyl-alt-4,7-bis(3-hexylthien-5-yl)-2,1,3-benzothiadiazole-2′,2′′-diyl) (F8TBT)/ poly(3-hexylthiophene) (P3HT) films are among the best polymer−polymer blend devices reported to date with a power conversion efficiency (PCE, ηe) of 1.9%.
In droplet-based microfluidics, non-ionic, high-molecular weight surfactants are required to stabilize droplet interfaces. One of the most common structures that imparts stability as well as ...biocompatibility to water-in-oil droplets is a triblock copolymer surfactant composed of perfluoropolyether (PFPE) and polyethylene glycol (PEG) blocks. However, the fast growing applications of microdroplets in biology would benefit from a larger choice of specialized surfactants. PEG as a hydrophilic moiety, however, is a very limited tool in surfactant modification as one can only vary the molecular weight and chain-end functionalization. In contrast, linear polyglycerol offers further side-chain functionalization to create custom-tailored, biocompatible droplet interfaces. Herein, we describe the synthesis and characterization of polyglycerol-based triblock surfactants with tailored side-chain composition, and exemplify their application in cell encapsulation and in vitro gene expression studies in droplet-based microfluidics.
Abstract
Cell-free protein synthesis has been widely used as a “breadboard” for design of synthetic genetic networks. However, due to a severe lack of modularity, forward engineering of genetic ...networks remains challenging. Here, we demonstrate how a combination of optimal experimental design and microfluidics allows us to devise dynamic cell-free gene expression experiments providing maximum information content for subsequent non-linear model identification. Importantly, we reveal that applying this methodology to a library of genetic circuits, that share common elements, further increases the information content of the data resulting in higher accuracy of model parameters. To show modularity of model parameters, we design a pulse decoder and bistable switch, and predict their behaviour both qualitatively and quantitatively. Finally, we update the parameter database and indicate that network topology affects parameter estimation accuracy. Utilizing our methodology provides us with more accurate model parameters, a necessity for forward engineering of complex genetic networks.
Type I interferon (IFN) is a key driver of immunity to infections and cancer. Plasmacytoid dendritic cells (pDCs) are uniquely equipped to produce large quantities of type I IFN but the mechanisms ...that control this process are poorly understood. Here we report on a droplet-based microfluidic platform to investigate type I IFN production in human pDCs at the single-cell level. We show that type I IFN but not TNFα production is limited to a small subpopulation of individually stimulated pDCs and controlled by stochastic gene regulation. Combining single-cell cytokine analysis with single-cell RNA-seq profiling reveals no evidence for a pre-existing subset of type I IFN-producing pDCs. By modulating the droplet microenvironment, we demonstrate that vigorous pDC population responses are driven by a type I IFN amplification loop. Our study highlights the significance of stochastic gene regulation and suggests strategies to dissect the characteristics of immune responses at the single-cell level.
Life is sustained by complex systems operating far from equilibrium and consisting of a multitude of enzymatic reaction networks. The operating principles of biology's regulatory networks are known, ...but the in vitro assembly of out-of-equilibrium enzymatic reaction networks has proved challenging, limiting the development of synthetic systems showing autonomous behaviour. Here, we present a strategy for the rational design of programmable functional reaction networks that exhibit dynamic behaviour. We demonstrate that a network built around autoactivation and delayed negative feedback of the enzyme trypsin is capable of producing sustained oscillating concentrations of active trypsin for over 65 h. Other functions, such as amplification, analog-to-digital conversion and periodic control over equilibrium systems, are obtained by linking multiple network modules in microfluidic flow reactors. The methodology developed here provides a general framework to construct dissipative, tunable and robust (bio)chemical reaction networks.
Conventional 2D cell culture techniques have provided fundamental insights into key biochemical and biophysical mechanisms responsible for various cellular behaviors, such as cell adhesion, ...spreading, division, proliferation, and differentiation. However, 2D culture in vitro does not fully capture the physical and chemical properties of the native microenvironment. There is a growing body of research that suggests that cells cultured on 2D substrates differ greatly from those grown in vivo. This article focuses on recent progress in using bioinspired 3D matrices that recapitulate as many aspects of the natural extracellular matrix as possible. A range of techniques for the engineering of 3D microenvironment with precisely controlled biophysical and chemical properties, and the impact of these environments on cellular behavior, is reviewed. Finally, an outlook on future challenges for engineering the 3D microenvironment and how such approaches would further our understanding of the influence of the microenvironment on cell function is provided.
Cells grown on 2D substrates differ dramatically from those grown in vivo and thus the 3D character of the microenvironment is required for development of many critical cell responses observed in vivo. Recent advances in the use of 3D microenvironments for cell biology research are described along with an overview of techniques for the fabrication of 3D microenvironments.
Kinetic modeling of in vitro enzymatic reaction networks is vital to understand and control the complex behaviors emerging from the nonlinear interactions inside. However, modeling is severely ...hampered by the lack of training data. Here, we introduce a methodology that combines an active learning-like approach and flow chemistry to efficiently create optimized datasets for a highly interconnected enzymatic reactions network with multiple sub-pathways. The optimal experimental design (OED) algorithm designs a sequence of out-of-equilibrium perturbations to maximize the information about the reaction kinetics, yielding a descriptive model that allows control of the output of the network towards any cost function. We experimentally validate the model by forcing the network to produce different product ratios while maintaining a minimum level of overall conversion efficiency. Our workflow scales with the complexity of the system and enables the optimization of previously unobtainable network outputs.
The incorporation of orthophosphate from scarce geochemical sources into the organic compounds essential for life under mild conditions is a fundamental challenge for prebiotic chemistry. Here we ...report a prebiotic system capable of overcoming this challenge by taking inspiration from extant life's recycling of orthophosphate via its conversion into kinetically stable thermodynamically activated (KSTA) nucleotide triphosphates (e.g. ATP). We separate the activation of orthophosphate from its transfer to organic compounds by, crucially, first accumulating a KSTA phosphoramidate. We use cyanate to activate orthophosphate in aqueous solution under mild conditions and then react it with imidazole to accumulate the KSTA imidazole phosphate. In a paste, imidazole phosphate phosphorylates all the essential building blocks of life. Integration of this chemistry into a wet/dry cycle enables the continuous recycling of orthophosphate and the accretion of phosphorylated compounds. This system functions even at low reagent concentrations due to solutes concentrating during evaporation. Our system demonstrates a general strategy for how to maximise the usage of scarce resources based upon cycles which accumulate and then release activated intermediates.
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