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Recent efforts in synthetic biology have shown the possibility of engineering distributed functions in populations of living cells, which requires the development of highly orthogonal, genetically encoded communication pathways. Cell-free transcription-translation (TXTL) reactions encapsulated in microcompartments enable prototyping of molecular communication channels and their integration into engineered genetic circuits by mimicking critical cell features, such as gene expression, cell size, and cell individuality within a community. In this review, we discuss the uses of cell-free transcription-translation reactions for the development of synthetic genetic circuits, with a special focus on the use of microcompartments supporting this reaction. We highlight several studies where molecular communication between non-living microcompartments and living cells have been successfully engineered., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The creation of adaptive matter is heavily inspired by biological systems. However, it remains challenging to design complex material responses that are governed by reaction networks, which lie at the heart of cellular complexity. The main reason for this slow progress is the lack of a general strategy to integrate reaction networks with materials. Herein we use a systematic approach to preprogram the response of a hydrogel to a trigger, in this case the enzyme trypsin, which activates a reaction network embedded within the hydrogel. A full characterization of all the kinetic rate constants in the system enabled the construction of a computational model, which predicted different hydrogel responses depending on the input concentration of the trigger. The results of the simulation are in good agreement with experimental findings. Our methodology can be used to design new, adaptive materials of which the properties are governed by reaction networks of arbitrary complexity., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Vesosomes are nested liposomal structures with high potential as advanced drug delivery vehicles, bioreactors and artificial cells. However, to date no method has been reported to prepare monodisperse vesosomes of controlled size. Here we report on a multistep microfluidic strategy for hierarchically assembling uniform vesosomes from dewetting of double emulsion templates. The control afforded by our method is illustrated by the formation of concentric, pericentric and multicompartment liposomes. The microfluidic route to vesosomes offers an exceptional platform to build artificial cells as exemplified by the in vitro transcription in "nucleus" liposomes and the mimicry of the architecture of eukaryotic cells. Finally, we show the transport of small molecules across the nucleic envelope via insertion of nanopores into the bilayers.

Secondary structure formation of mRNA, caused by desynchronization of transcription and translation, is known to impact gene expression in vivo. Yet, inactivation of mRNA by secondary structures in cell-free protein expression is frequently overlooked. Transcription and translation rates are often not highly synchronized in cell-free expression systems, leading to a temporal mismatch between the processes and a drop in efficiency of protein production. By devising a cell-free gene expression platform in which transcriptional and translational elongation are successfully performed independently, we determine that sequence-dependent mRNA secondary structures are the main cause of mRNA inactivation in in vitro gene expression.

In recent years, there is a growing interest in designing artificial analogues of living systems, fueled not only by potential applications as 'smart micro-machines', but also by the demand for simple models that can be used to study the behavior of their more complex natural counterparts. Here, we present a facile, internally driven, experimental system comprised of fluorescently labeled colloidal silica rods of which the self-propulsion is powered by the decomposition of H 2 O 2 catalyzed by a length-wise half Pt coating of the particles in order to study how shape anisotropy and swimming direction affect the collective behavior. We investigated the emerging structures and their time evolution for various particle concentrations in (quasi-)two dimensional systems for three aspect ratios of the rods on a single particle level using a combination of experiments and simulations. We found that the dynamic self-organization relied on a competition between self-propulsion and phoretic attractions induced by phoresis of the rods. We observed that the particle clustering behavior depends on the concentration as well as the aspect ratio of the rods. Our findings provide a more detailed understanding of dynamic self-organization of anisotropic particles and the role the propulsion direction plays in internally driven systems.

Every living cell is a compartmentalized out-of-equilibrium system exquisitely able to convert chemical energy into function. In order to maintain homeostasis, the flux of metabolites is tightly controlled by regulatory enzymatic networks. A crucial prerequisite for the development of lifelike materials is the construction of synthetic systems with compartmentalized reaction networks that maintain out-of-equilibrium function. Here, we aim for autonomous movement as an example of the conversion of feedstock molecules into function. The flux of the conversion is regulated by a rationally designed enzymatic reaction network with multiple feedforward loops. By compartmentalizing the network into bowl-shaped nanocapsules the output of the network is harvested as kinetic energy. The entire system shows sustained and tunable microscopic motion resulting from the conversion of multiple external substrates. The successful compartmentalization of an out-of-equilibrium reaction network is a major first step in harnessing the design principles of life for construction of adaptive and internally regulated lifelike systems.

The number of circulating tumor cells (CTCs) in blood is strongly correlated with the progress of metastatic cancer. Current methods to detect CTCs are based on immunostaining or discrimination of physical properties. Herein, a label-free method is presented exploiting the abnormal metabolic behavior of cancer cells. A single-cell analysis technique is used to measure the secretion of acid from individual living tumor cells compartmentalized in microfluidically prepared, monodisperse, picoliter (pL) droplets. As few as 10 tumor cells can be detected in a background of 200 000 white blood cells and proof-of-concept data is shown on the detection of CTCs in the blood of metastatic patients., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

For some decades now, nanotechnology has been touted as the 'next big thing' with potential impact comparable to the steam, electricity or Internet revolutions - but has it lived up to these expectations? While advances in top-down nanolithography, now reaching 10-nm resolution, have resulted in devices that are rapidly approaching mass production, attempts to produce nanoscale devices using bottom-up approaches have met with only limited success. We have been inundated with nanoparticles of almost any shape, material and composition, but their societal impact has been far from revolutionary, with growing concerns over their toxicity. Despite nebulous hopes that making hierarchical nanomaterials will lead to new, emergent properties, no breakthrough applications seem imminent. In this Perspective, we argue that the time is ripe to look beyond individual nano-objects and their static assemblies, and instead focus on systems comprising different types of 'nanoparts' interacting and/or communicating with one another to perform desired functions. Such systems are interesting for a variety of reasons: they can act autonomously without external electrical or optical connections, can be dynamic and reconfigurable, and can act as 'nanomachines' by directing the flow of mass, energy or information . In thinking how this systems nanoscience approach could be implemented to design useful - as opposed to toy-model - nanosystems, our choice of applications and our nanoengineering should be inspired by living matter.

Liposomes are self-assembled phospholipid vesicles with great potential in fields ranging from targeted drug delivery to artificial cells. The formation of liposomes using microfluidic techniques has seen considerable progress, but the liposomes formation process itself has not been studied in great detail. As a result, high throughput, high-yielding routes to monodisperse liposomes with multiple compartments have not been demonstrated. Here, we report on a surfactant-assisted microfluidic route to uniform, single bilayer liposomes, ranging from 25 to 190 μm, and with or without multiple inner compartments. The key of our method is the precise control over the developing interfacial energies of complex W/O/W emulsion systems during liposome formation, which is achieved via an additional surfactant in the outer water phase. The liposomes consist of single bilayers, as demonstrated by nanopore formation experiments and confocal fluorescence microscopy, and they can act as compartments for cell-free gene expression. The microfluidic technique can be expanded to create liposomes with a multitude of coupled compartments, opening routes to networks of multistep microreactors.

In recent years, technologies capable of analyzing single cells have emerged that are transforming many fields of biological research. Herein we report how DNA-functionalized hydrogel beads can serve as a matrix to capture mRNA from lysed single cells. mRNA quantification free of pre-amplification bias is ensured by using padlock probes and rolling circle amplification followed by hybridization with fluorescent probes. The number of transcripts in individual cells is assessed by simply counting fluorescent dots inside gel beads. The method extends the potential of existing techniques and provides a general platform for capturing molecules of interest from single cells., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Herein, we describe a simple and robust approach to repeatedly modify surfaces with polymer brushes through surface-initiated atomic transfer radical polymerization (SI-ATRP), based on an initiator-embedded polystyrene sheet that does not rely on specific surface chemistries for initiator immobilization. The surface-grafted polymer brushes can be wiped away to expose fresh underlying initiator that re-initiates polymerization. This strategy provides a facile route for modification of molded or embossed surfaces, with possible applications in the preparation of fluidic devices and polymer-embedded circuits., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In highly crowded and viscous intracellular environments, the kinetics of complex enzymatic reactions are determined by both reaction and diffusion rates. However in vitro studies on transcription and translation often fail to take into account the density of the prokaryotic cytoplasm. Here we mimic the cellular environment by using a porous hydrogel matrix, to study the effects of macromolecular crowding on gene expression. We found that within microgels gene expression is localized, transcription is enhanced up to fivefold, and translation is enhanced up to fourfold. Our results highlight the need to consider the role of the physical environment on complex biochemical reactions, in this case macromolecular crowding, nanoscale spatial organization, and confinement., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Understanding the dynamics of complex enzymatic reactions in highly crowded small volumes is crucial for the development of synthetic minimal cells. Compartmentalized biochemical reactions in cell-sized containers exhibit a degree of randomness due to the small number of molecules involved. However, it is unknown how the physical environment contributes to the stochastic nature of multistep enzymatic processes. Here, we present a robust method to quantify gene expression noise in vitro using droplet microfluidics. We study the changes in stochasticity in the cell-free gene expression of two genes compartmentalized within droplets as a function of DNA copy number and macromolecular crowding. We find that decreased diffusion caused by a crowded environment leads to the spontaneous formation of heterogeneous microenvironments of mRNA as local production rates exceed the diffusion rates of macromolecules. This heterogeneity leads to a higher probability of the molecular machinery staying in the same microenvironment, directly increasing the system's stochasticity.

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.

Water-in-oil-in-water (w/o/w) microfluidics double emulsions offer a new route to compartmentalise reagents into isolated aqueous microenvironments while maintaining an aqueous carrier fluid phase; this enables compatibility with commercial flow cytometry systems such as fluorescence-activated cell sorting (FACS). Double emulsion (inner core) deformation under hydrodynamic focusing conditions that mimic the environment double emulsions experience in flow cytometry applications is of particular importance for droplet stability and cell viability. This paper reports on an experimental study of the dynamic deformation of aqueous cores of w/o/w double emulsions under hydrodynamic focusing, with the sheath flow directed at 45° to the sample flow. A number of factors affecting the inner core deformation and recovery were examined. Deformation was found to depend significantly on the core or shell viscosity, the droplet-to-sheath flow velocity ratio, and core and shell sizes. Core deformation was found to depend more on the type of surfactant rather concentration with high molecular weight surfactant exhibiting a negligible effect on deformation whereas low molecular weight surfactant enhancing deformation at low concentrations due to their lateral mobility at the interface.

Living cells are able to produce a wide variety of biological responses when subjected to biochemical stimuli. It has become apparent that these biological responses are regulated by complex chemical reaction networks (CRNs). Unravelling the function of these circuits is a key topic of both systems biology and synthetic biology. Recent progress at the interface of chemistry and biology together with the realisation that current experimental tools are insufficient to quantitatively understand the molecular logic of pathways inside living cells has triggered renewed interest in the bottom-up development of CRNs. This builds upon earlier work of physical chemists who extensively studied inorganic CRNs and showed how a system of chemical reactions can give rise to complex spatiotemporal responses such as oscillations and pattern formation. Using purified biochemical components, in vitro synthetic biologists have started to engineer simplified model systems with the goal of mimicking biological responses of intracellular circuits. Emulation and reconstruction of system-level properties of intracellular networks using simplified circuits are able to reveal key design principles and molecular programs that underlie the biological function of interest. In this Tutorial Review, we present an accessible overview of this emerging field starting with key studies on inorganic CRNs followed by a discussion of recent work involving purified biochemical components. Finally, we review recent work showing the versatility of programmable biochemical reaction networks (BRNs) in analytical and diagnostic applications.

The cytosol of Escherichia coli is an extremely crowded environment, containing high concentrations of biopolymers which occupy 20-30% of the available volume. Such conditions are expected to yield depletion forces, which strongly promote macromolecular complexation. However, crowded macromolecule solutions, like the cytosol, are very prone to nonspecific associative interactions that can potentially counteract depletion. It remains unclear how the cytosol balances these opposing interactions. We used a FRET-based probe to systematically study depletion in vitro in different crowded environments, including a cytosolic mimic, E. coli lysate. We also studied bundle formation of FtsZ protofilaments under identical crowded conditions as a probe for depletion interactions at much larger overlap volumes of the probe molecule. The FRET probe showed a more compact conformation in synthetic crowding agents, suggesting strong depletion interactions. However, depletion was completely negated in cell lysate and other protein crowding agents, where the FRET probe even occupied slightly more volume. In contrast, bundle formation of FtsZ protofilaments proceeded as readily in E. coli lysate and other protein solutions as in synthetic crowding agents. Our experimental results and model suggest that, in crowded biopolymer solutions, associative interactions counterbalance depletion forces for small macromolecules. Furthermore, the net effects of macromolecular crowding will be dependent on both the size of the macromolecule and its associative interactions with the crowded background.

Our knowledge of the properties and dynamics of complex molecular reaction networks, for example those found in living systems, considerably lags behind the understanding of elementary chemical reactions. In part, this is because chemical reactions networks are nonlinear systems that operate under conditions far from equilibrium. Of particular interest is the role of individual reaction rates on the stability of the network output. In this research we use a rational approach combined with computational methods, to produce complex behavior (in our case oscillations) and show that small changes in molecular structure are sufficient to impart large changes in network behavior.

The flow environment in inner cores of water-in-oil-in-water (w/o/w) microfluidic double emulsions has a significant impact on industrial applications of such systems. For example, in the case of shear sensitive cells compartmentalised in the cores, high shear conditions may be deleterious. This study reports on the flow characteristics of w/o/w inner cores in comparison to those in single water-in-oil (w/o) microdroplets of equal size moving in the same microchannel, resolved by means of micro-particle image velocimetry (μPIV). The multiphase flow system employed in the study had a viscosity ratio, λ, between aqueous and oil phase of the order of unity (λ = 0.78) and both single droplets and inner cores of double emulsions filled the channel. This configuration resulted in a weak recirculating flow inside the w/o single droplet: the measured flow field exhibited a uniform low velocity flow field in the central region surrounded by small regions of reversed flow near the channel walls. This flow topology was maintained in the inner cores of w/o/w double emulsions for intermediate capillary numbers (Ca) ranging from 10(-3) to 10(-2), and core morphologies varying from large plugs to pancake cores. The core morphology affected the magnitude and distribution of the velocity in the droplets. The similarity in the flow topology resulted from the fact that inner cores were located at the back of the outer droplet in such a way that inner and outer interfaces were in contact for over half of core surface area and separated only by a thin lubricating film. Both single droplets and inner cores exhibited a narrow shear rate distribution characterised by small regions of maximum shear confined near the channel walls. Shear rate magnitude values were found to be an order of magnitude lower than those in the channel and hence capable of reducing stresses in flow cytometry to far below reported values for cell damage. Hence, it can be concluded that double emulsions are suitable candidates to substitute single droplets in flow cytometry to protect the screened items and are compatible with the commercial flow cytometry systems.

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.

The flow topology in moving microdroplets has a significant impact on the behaviour of encapsulated objects and hence on applications of the technology. This study reports on a systematic investigation of the flow field inside droplets moving in a rectangular microchannel, by means of micro-particle image velocimetry (μPIV). Various water/oil (w/o) fluid mixtures were studied in order to elucidate the effects of a number of parameters such as capillary number (Ca), droplet geometry, viscosity ratio and interfacial tension. A distinct change in flow topology was observed at intermediate Ca ranging from 10(-3) to 10(-1), in surfactant-laden droplets, which was attributed primarily to the viscosity ratio of the two phases rather than the Marangoni effect expected in such systems. W/o droplet systems of lower inner-to-outer viscosity ratios tend to exhibit the well-known flow pattern characterised by a parabola-like profile in the droplet bulk-volume, surrounded by two counter rotating recirculation zones on either side of the droplet axis. As the viscosity ratio between the two phases is increased, the flow pattern becomes more uniform, exhibiting low velocities in the droplet bulk-volume and higher-reversed velocities along the w/o interface. The Ca and droplet geometry had no effect on the observed flow topology change. The study highlights the complex, three-dimensional (3D) nature of the flow inside droplets in rectangular microchannels and demonstrates the ability to control the droplet flow environment by adjusting the viscosity ratio between the two phases.

Mechanically strong supramolecular hydrogels (up to 98.9% water content) were obtained by the combination of a rigid, fully π-conjugated polymer backbone and zwitterionic side chains. The gels were characterized by SAXS, SEM and rheology measurements and are fluorescent, stimuli responsive (temperature, salts) and bind DNA.

We microfluidically fabricate bio-orthogonal DNA-functionalized porous hydrogels from hyaluronic acid that are employed in in vitro transcription/translation (IVTT) of a green fluorescent protein. By co-encapsulating individual hydrogel particles and the IVTT machinery in water-in-oil microdroplets, we study protein expression in a defined reaction volume. Our approach enables precise control over protein expression rates by gene dosage. We show that gene transcription and translation are confined to the membrane-free hydrogel matrix, which contributes to the design of membrane-free protocells.

A wet stamping method to precisely control concentrations of enzymes and inhibitors in place and time inside layered gels is reported. By combining enzymatic reactions such as autocatalysis and inhibition with spatial delivery of components through soft lithographic techniques, a biochemical reaction network capable of recognizing the spatial distribution of an enzyme was constructed. The experimental method can be used to assess fundamental principles of spatiotemporal order formation in chemical reaction networks., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Droplet-based microfluidics is increasingly used for biological applications, where the recovery of cells or particles after an experiment or assay is desirable. Here, we present an electro-demulsification chip which circumvents the use of harsh chemicals and multiple washing/centrifugation steps and offers a mild way for extracting cells and polymer particles into an aqueous phase from microfluidic water-in-oil emulsions.

Here, we report a convenient experimental platform to study the diffusion of Ca(2+) in the presence of a Ca(2+)-binding protein (Calbindin D28k). This work opens up new possibilities to elucidate the physical chemistry of complex Ca(2+)-dependent reaction-diffusion networks that are abundant in living cells.

Cell culturing, whether for tissue engineering or cell biology studies, always involves placing cells in a non-natural environment and no material currently exist that can mimic the entire complexity of natural tissues and variety of cell-matrix interactions that is found in vivo. Here, we review the vast range of hydrogels, composed of natural or synthetic polymers that provide a route to tailored microenvironments., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A series of water-soluble macro-initiators is synthesized to avoid radical loss in microfluidic on-chip photo cross-linking of hyaluronic acid methacrylate-containing water-in-oil emulsions. Their superior performance over known photo-initiators through the generation of water-soluble radicals and excellent biocompatibility are demonstrated.

Red blood cells (RBCs) undergo extensive deformation when travelling through the microcapillaries. Deformability, the combined result of properties of the membrane-cytoskeleton complex, the surface area-to-volume ratio, and the hemoglobin content, is a critical determinant of capillary blood flow. During blood bank storage and in many pathophysiological conditions, RBC morphology changes, which has been suggested to be associated with decreased deformability and removal of RBC. While various techniques provide information on the rheological properties of stored RBCs, their clinical significance is controversial. We developed a microfluidic approach for evaluating RBC deformability in a physiologically meaningful and clinically significant manner. Unlike other techniques, our method enables a high-throughput determination of changes in deformation capacity to provide statistically significant data, while providing morphological information at the single-cell level. Our data show that, under conditions that closely mimic capillary dimensions and flow, the capacity to deform and the capacity to relax are not affected during storage in the blood bank. Our data also show that altered cell morphology by itself does not necessarily affect deformability.

Here, we present a platform to detect cytokine (IL-2, IFN-γ, TNF-α) secretion of single, activated T-cells in droplets over time. We use a novel droplet-based microfluidic approach to encapsulate cells in monodisperse agarose droplets together with functionalized cytokine-capture beads for subsequent binding and detection of secreted cytokines from single cells. This method allows high-throughput detection of cellular heterogeneity and maps subsets within cell populations with specific functions.

Delineating design principles of biological systems by reconstitution of purified components offers a platform to gauge the influence of critical physicochemical parameters on minimal biological systems of reduced complexity. Here we unravel the effect of strong reversible inhibitors on the spatiotemporal propagation of enzymatic reactions in a confined environment in vitro. We use micropatterned, enzyme-laden agarose gels which are stamped on polyacrylamide films containing immobilized substrates and reversible inhibitors. Quantitative fluorescence imaging combined with detailed numerical simulations of the reaction-diffusion process reveal that a shallow gradient of enzyme is converted into a steep product gradient by addition of strong inhibitors, consistent with a mathematical model of molecular titration. The results confirm that ultrasensitive and threshold effects at the molecular level can convert a graded input signal to a steep spatial response at macroscopic length scales., (Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The field of stem cells and regenerative medicine offers considerable promise as a means of delivering new treatments for a wide range of diseases. In order to maximize the effectiveness of cell-based therapies - whether stimulating expansion of endogenous cells or transplanting cells into patients - it is essential to understand the environmental (niche) signals that regulate stem cell behaviour. One of those signals is from the extracellular matrix (ECM). New technologies have offered insights into how stem cells sense signals from the ECM and how they respond to these signals at the molecular level, which ultimately regulate their fate.

We report a microfluidic droplet-based approach enabling the measurement of chemical reactions of individual enzyme molecules and its application to a single-molecule-counting immunoassay. A microfluidic device is used to generate and manipulate <10 fL droplets at rates of up to 1.3 × 10(6) per second, about 2 orders of magnitude faster than has previously been reported. The femtodroplets produced with this device can be used to encapsulate single biomolecular complexes tagged with a reporter enzyme; their small volume enables the fluorescent product of a single enzyme molecule to be detected within 10 min of on-chip incubation. Our prototype system is validated by detection of a biomarker for prostate cancer in buffer, down to a concentration of 46 fM. This work demonstrates a highly flexible and sensitive diagnostic platform that exploits extremely high-speed generation of monodisperse femtoliter droplets for the counting of individual analyte molecules.

Liquid-liquid phase transitions in complex mixtures of proteins and other molecules produce crowded compartments supporting in vitro transcription and translation. We developed a method based on picoliter water-in-oil droplets to induce coacervation in Escherichia coli cell lysate and follow gene expression under crowded and noncrowded conditions. Coacervation creates an artificial cell-like environment in which the rate of mRNA production is increased significantly. Fits to the measured transcription rates show a two orders of magnitude larger binding constant between DNA and T7 RNA polymerase, and five to six times larger rate constant for transcription in crowded environments, strikingly similar to in vivo rates. The effect of crowding on interactions and kinetics of the fundamental machinery of gene expression has a direct impact on our understanding of biochemical networks in vivo. Moreover, our results show the intrinsic potential of cellular components to facilitate macromolecular organization into membrane-free compartments by phase separation.

The intricacy of the different parameters involved in cell adhesion to biomaterials and fate decision (e.g. proliferation, differentiation, apoptosis) makes the decoupling of the respective effects of surface properties, extra-cellular matrix protein adsorption and ultimately cell behaviour difficult. This work presents a micro-patterned polymer brush platform to control the adsorption of extra-cellular matrix (ECM) proteins to well defined micron-size areas and consequently control cell adhesion, spreading and shape independently of other chemical and physical surface properties. Protein patterns can be readily generated with brushes presenting a range of hydrophilicity and surface charge density. The surface properties of the selected brushes are fully characterised using a combination of FTIR, XPS, ellipsometry, atomic force microscopy, water contact goniometry, dynamic light scattering and ζ-potential measurements. Interactions of proteins relevant to cell patterning and culture with these brushes are studied by surface plasmon resonance, dynamic light scattering, ellipsometry and immuno-fluorescence microscopy. Finally this platform is used in an assay investigating the relative contributions of matrix geometry and surface chemistry on epidermal stem cell differentiation. It is found that moderate hydrophobicity does not impact stem cell commitment, whereas strongly negative surface potential increases the incidence of differentiation. This correlates with a marked decrease in the formation of focal adhesions (but not cell spreading).

In this paper, we investigated the intra-species bacterial quorum sensing at the single cell level using a double droplet trapping system. Escherichia coli transformed to express the quorum sensing receptor protein, LasR, were encapsulated in microdroplets that were positioned adjacent to microdroplets containing the autoinducer, N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL). Functional activation of the LasR protein by diffusion of the OdDHL across the droplet interface was measured by monitoring the expression of green fluorescent protein (GFP) from a LasR-dependent promoter. A threshold concentration of OdDHL was found to induce production of quorum-sensing associated GFP by E. coli. Additionally, we demonstrated that LasR-dependent activation of GFP expression was also initiated when the adjacent droplets contained single E. coli transformed with the OdDHL synthase gene, LasI, representing a simple quorum sensing circuit between two droplets.

Nonspecific protein adsorption is a central challenge for the use of polymeric materials in biological media. While the quantity of adsorbed protein can be lowered, very few surfaces are protein resistant when exposed to undiluted serum or plasma. The underlying principles of this fouling and the adsorbing proteins remain to be identified. Here, we investigated adsorption from undiluted human blood plasma to three different polymer brushes. Our study showed that the polymer structure does not influence which proteins adsorb. Further, we identified 98 plasma proteins that still foul current "protein-resistant" polymer brushes. Detailed studies into the major adsorbing protein revealed the central role that lipoproteins and low density lipoprotein in particular play in fouling of plasma to polymeric biomaterials. However, although apolipoprotein B100 is found as a major fouling protein in our mass spectrometry screening, studies on individual components of lipoproteins show that it is not apoB100 but a mixture of phospholipids, triglycerides, and cholesteryl esters that plays a major role in lipoprotein adsorption.

We report the electronic properties of the conjugated coupling between a donor polymer and an acceptor segment serving as a model for the coupling in conjugated donor-acceptor block copolymers. These structures allow the study of possible intrachain photoinduced charge separation, in contrast to the interchain separation achieved in conventional donor-acceptor blends. Depending on the nature of the conjugated linkage, we observe varying degrees of modification of the excited states, including the formation of intrachain charge transfer excitons. The polymers comprise a block (typically 18 repeat units) of P3HT, poly(3-hexyl thiophene), coupled to a single unit of F8-TBT (where F8 is dioctylfluorene, and TBT is thiophene-benzothiadiazole-thiophene). When the P3HT chain is linked to the TBT unit, we observe formation of a localized charge transfer state, with red-shifted absorption and emission. Independent of the excitation energy, this state is formed very rapidly (<40 fs) and efficiently. Because there is only a single TBT unit present, there is little scope for long-range charge separation and it is relatively short-lived, <1 ns. In contrast, when the P3HT chain and TBT unit are separated by the wider bandgap F8 unit, there is little indication for modification of either ground or excited electronic states, and longer-lived charge separated states are observed.

There has been phenomenal effort synthesizing new low-band gap polymer hole-conductors which absorb into the near-infrared (NIR), leading to >10% efficient all-organic solar cells. However, organic light absorbers have relatively narrow bandwidths, making it challenging to obtain panchromatic absorption in a single organic semiconductor. Here, we demonstrate that (poly[2,6-(4,4-bis-(2-ethylhexyl)-4H-cyclopenta[2,1-b;3,4-b0]dithiophene)-alt-4,7-(2,1,3-benzothiadia-zole)] (PCPDTBT) can be "photo-sensitized" across the whole visible spectrum by "doping" with a visible absorbing dye, the (2,2,7,7-tetrakis(3-hexyl-5-(7-(4-hexylthiophen-2-yl)benzo[c][1,2,5]thiadiazol-4-yl)thiophen-2-yl)-9,9-spirobifluorene) (spiro-TBT). Through a comprehensive sub-12 femtosecond-nanosecond spectroscopic study, we demonstrate that extremely efficient and fast energy transfer occurs from the photoexcited spiro-TBT to the PCPDTBT, and ultrafast charge injection happens when the system is interfaced with ZnO as a prototypal electron-acceptor compound. The visible photosensitization can be effectively exploited and gives panchromatic photoresponse in prototype polymer/oxide bilayer photovoltaic diodes. This concept can be successfully adopted for tuning and optimizing the light absorption and photoresponse in a broad range of polymeric and hybrid solar cells.

Surface initiated atom transfer radical polymerization (SI-ATRP) was triggered after diffusion of a Cu(I)/L activator generated at a working electrode. A stable [Cu(II)L]/[Cu(I)L] ratio gradient was formed at the gap between the working electrode and the initiator terminated substrate due to ion diffusion. The size of the gap can be used to dictate polymer growth kinetics at different gap distances. Gradient polymer brushes were grafted when substrate was placed at a tilting angle along [Cu(II)L]/[Cu(I)L] gradient.

We have demonstrated capture and release of underwater-oil droplets based on fouling-resistant surfaces coated with pH-responsive polymer brushes. In response to the change of environmental pH, oil droplets were captured on the polymer brush-modified surfaces in the high adhesion state. As the droplet volume increased upon coalescence with other oil droplets in the aqueous phase, the captured droplets eventually self-released from the surfaces under the influence of buoyancy and rose to the air-water interface. The fact that the polymer brush surfaces were partially oil-wettable (high oil-in-water contact angles) enabled the adhesion but not the spreading of oil droplets. This allowed buoyancy release of oil droplets and led to fouling-resistant surfaces that could be reused for capture-release of more oil droplets. The practicality and versatility of this oil droplet capture-release system was demonstrated using monodisperse and polydisperse hydrocarbon oil compositions in purified water, tap water, and brines in which the salt concentration was as high as that of seawater.

We present a fast and efficient in situ synthetic approach to obtain fully π-conjugated polymers with degrees of polymerization up to 23 and near quantitative (>95%) heterobis-functionalization. The synthesis relies on the key advantages of controlled Suzuki chain-growth polymerization: control over molecular weight, narrow polydispersity, and ability to define polymer end groups. The first end group is introduced through the initiator metal complex tBu(3)PPd(X)Br, while the second end group is added by quenching of the chain-growth polymerization with the desired boronic esters. In all cases, polymers obtained at 50% conversion showed excellent end group fidelity and high purity following a simple workup procedure, as determined by MALDI-TOF, GPC, and (1)H and 2D NMR. End group functionalization altered the optoelectronic properties of the bridge polymer. Building on a common fluorene backbone, and guided by DFT calculations, we introduced donor and acceptor end groups to create polymeric molecular wires exhibiting charge transfer and energy transfer as characterized by fluorescence, absorption, and transient absorption spectroscopy as well as by fluorescence lifetime measurements.

Microgel particles are formed from aqueous-two-phase-system (ATPS) droplets in poly(dimethylsiloxane) (PDMS) microfluidic devices. The droplets consist of a dextran core and a photopolymerizable poly(ethylene glycol) diacrylate (PEGDA) shell. Upon UV exposure, the ATPS droplets undergo a shape-transformation yielding PEGDA microgel particles containing a socket., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Correct tissue architecture is essential for normal physiology, yet there have been few attempts to recreate tissues using micro-patterning. We have used polymer brush micro-engineering to generate a stratified micro-epidermis with fewer than 10 human keratinocytes. Epidermal stem cells are captured on 100 μm diameter circular collagen-coated disks. Within 24 h they assemble a stratified micro-tissue, in which differentiated cells have a central suprabasal location. For rings with a non-adhesive centre of up to 40 μm diameter, cell-cell and cell-matrix adhesive interactions together result in correct micro-epidermis assembly. Assembly requires actin polymerization, adherens junctions and desmosomes, but not myosin II-mediated contractility nor coordinated cell movement. Squamous cell carcinoma cells on micro-patterned rings exhibit disturbed architecture that correlates with the characteristics of the original tumours. The micro-epidermis we have generated provides a new platform for screening drugs that modulate tissue assembly, quantifying tissue stratification and investigating the properties of tumour cells., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

To investigate how substrate properties influence stem-cell fate, we cultured single human epidermal stem cells on polydimethylsiloxane (PDMS) and polyacrylamide (PAAm) hydrogel surfaces, 0.1 kPa-2.3 MPa in stiffness, with a covalently attached collagen coating. Cell spreading and differentiation were unaffected by polydimethylsiloxane stiffness. However, cells on polyacrylamide of low elastic modulus (0.5 kPa) could not form stable focal adhesions and differentiated as a result of decreased activation of the extracellular-signal-related kinase (ERK)/mitogen-activated protein kinase (MAPK) signalling pathway. The differentiation of human mesenchymal stem cells was also unaffected by PDMS stiffness but regulated by the elastic modulus of PAAm. Dextran penetration measurements indicated that polyacrylamide substrates of low elastic modulus were more porous than stiff substrates, suggesting that the collagen anchoring points would be further apart. We then changed collagen crosslink concentration and used hydrogel-nanoparticle substrates to vary anchoring distance at constant substrate stiffness. Lower collagen anchoring density resulted in increased differentiation. We conclude that stem cells exert a mechanical force on collagen fibres and gauge the feedback to make cell-fate decisions.