Prebiotic environments are dynamic, containing a range of periodic and aperiodic variations in reaction conditions. However, the impact of the temporal dynamics of environmental conditions upon prebiotic chemical reaction networks has not been investigated. Here, we demonstrate how the magnitude and rate of temporal fluctuations of the catalysts Ca2+ and hydroxide control the product distributions of the formose reaction. Surprisingly, the product compositions of the formose reaction under dynamic conditions deviate significantly from those under steady state conditions. We attribute these compositional changes to the non-uniform propagation of fluctuations through the network, thereby shaping reaction outcomes. An examination of temporal concentration patterns showed that collections of compounds responded collectively to perturbations, indicating that key gating reactions branching from the Breslow cycle may be important responsive features of the formose reaction. Our findings show how the compositions of prebiotic reaction networks were shaped by sequential environmental events, illustrating the necessity for considering the temporal traits of prebiotic environments that supported the origin of life.
Publications
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337. Dynamic environmental conditions affect the composition of a model Prebiotic Reaction Network
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336. Computing arithmetic functions using immobilised enzymatic Reaction Networks
Living systems use enzymatic reaction networks to process biochemical information and make decisions in response to external or internal stimuli. Herein, we present a modular and reusable platform for molecular information processing using enzymes immobilised in hydrogel beads and compartmentalised in a continuous stirred tank reactor. We demonstrate how this setup allows us to perform simple arithmetic operations, such as addition, subtraction and multiplication, using various concentrations of substrates or inhibitors as inputs and the production of a fluorescent molecule as the readout.
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335. Tuning material states and functionalities of G-quadruplex-modulated RNA-peptide condensates
RNA encodes sequence- and structure-dependent interactions to modulate the assembly and properties of biomolecular condensates. RNA G-quadruplexes (rG4s) formed by guanine-rich sequences can trigger the formation of liquid- or solid-like condensates that are involved in many aberrant phase transitions. However, exactly how rG4 motifs modulate different phase transitions and impart distinct material properties to condensates is unclear. Here, using RNA oligonucleotides and cationic peptides as model systems, we show that RNA-peptide condensates exhibit tunability in material properties over a wide spectrum via interactions arising from rG4 folding/unfolding kinetics. rG4-containing oligonucleotides formed strong pairwise attraction with peptides and tended to form solid-like condensates, while their less-structured non-G4 mutants formed liquid-like droplets. We find that the coupling between rG4 dissociation and RNApeptide complex coacervation triggers solid-to-liquid transition of condensates prior to the complete unfolding of rG4s. This coupling points to a mechanism that material states of rG4-modulated condensates can be finely tuned from solid-like to liquid-like by the addition of less-structured RNA oligonucleotides, which have weak but dominant binding with peptides. We further show that the tunable material states of condensates can enhance RNA aptamer compartmentalization and RNA cleavage reactions. Our results suggest that condensates with complex properties can emerge from subtle changes in RNA oligonucleotides, contributing ways to treat dysfunctional condensates in diseases and insights into prebiotic compartmentalization.
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334. Integrated single-cell (phospho-)protein and RNA detection uncovers phenotypic characteristics and active signal transduction of human antibody-secreting cells
Single-cell technologies are currently widely applied to obtain a deeper understanding of the phenotype of single-cells in heterogenous mixtures. However, integrated multilayer approaches including simultaneous detection of mRNA, protein expression, and intracellular phospho-proteins are still challenging. Here, we combined an adapted method to in vitrodifferentiate peripheral B-cells into antibody-secreting cells (ASCs) (i.e., plasmablasts and plasma cells) with integrated multi-omic single-cell sequencing technologies to detect and quantify immunoglobulin subclass-specific surface markers, transcriptional profiles, and signaling transduction pathway components. Using a common set of surface proteins, we integrated two multimodal datasets to combine mRNA, protein expression, and phospho-protein detection in one integrated dataset. Next, we tested whether ASCs that only seem to differ in its ability to secrete different IgM, IgA, or IgG antibodies exhibit other differences that characterize these different ASCs. Our approach detected differential expression of plasmablast and plasma cell markers, homing receptors, and TNF receptors. In addition, differential sensitivity was observed for the different cytokine stimulations that were applied during in vitro differentiation. For example, IgM ASCs were more sensitive to IL-15, while IgG ASC responded more to IL-6 and IFN addition. Furthermore, tonic BCR activity was detected in IgA and IgM ASCs, while IgG ASC exhibited active BCR-independent SYK activity and NF-?B and mTOR signaling. We confirmed these findings using flow cytometry and small molecules inhibitors, demonstrating the importance of SYK, NF-?B, and mTOR activity for plasmablast/plasma cell differentiation/survival and/or IgG secretion. Taken together, our integrated multi-omics approach allowed high-resolution phenotypic characterization of single cells in a heterogenous sample of in vitrodifferentiated human ASCs. Our strategy is expected to further our understanding of human ASCs in healthy and diseased samples and provide a valuable tool to identify novel biomarkers and potential drug targets.
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333. Strategies for transferring photobiocatalysis to continuous flow exemplified by photodecarboxylation of fatty acids
The challenges of light-dependent biocatalytic transformations of lipophilic substrates in aqueous media are manifold. For instance, photolability of the catalyst as well as insufficient light penetration into the reaction vessel may be further exacerbated by a heterogeneously dispersed substrate. Light penetration may be addressed by performing the reaction in continuous flow, which allows two modes of applying the catalyst: (i) heterogeneously, immobilized on a carrier, which requires light-permeable supports, or (ii) homogeneously, dissolved in the reaction mixture. Taking the light-dependent photodecarboxylation of palmitic acid catalyzed by fatty-acid photodecarboxylase from Chlorella variabilis (CvFAP) as a showcase, strategies for the transfer of a photoenzyme-catalyzed reaction into continuous flow were identified. A range of different supports were evaluated for the immobilization of CvFAP, whereby Eupergit C250 L was the carrier of choice. As the photostability of the catalyst was a limiting factor, a homogeneous system was preferred instead of employing the heterogenized enzyme. This implied that photolabile enzymes may preferably be applied in solution if repair mechanisms cannot be provided. Furthermore, when comparing different wavelengths and light intensities, extinction coefficients may be considered to ensure comparable absorption at each wavelength. Employing homogeneous conditions in the CvFAP-catalyzed photodecarboxylation of palmitic acid afforded a space-time yield unsurpassed by any reported batch process (5.7 g·L1·h1, 26.9 mmol·L1·h1) for this reaction, demonstrating the advantage of continuous flow in attaining higher productivity of photobiocatalytic processes.
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332. Effective screening of coulomb repulsions in water accelerates reactions of like-charged compounds by orders of magnitude
The reaction kinetics between like-charged compounds in water is extremely slow due to Coulomb repulsions. Here, we demonstrate that by screening these interactions and, in consequence, increasing the local concentration of reactants, we boost the reactions by many orders of magnitude. The reaction between negatively charged Coenzyme A molecules accelerates ~5 million-fold using cationic micelles. That is ~104 faster kinetics than in 0.5 M NaCl, although the salt is ~106 more concentrated. Rate enhancements are not limited to micelles, as evidenced by significant catalytic effects (104105-fold) of other highly charged species such as oligomers and polymers. We generalize the observed phenomenon by analogously speeding up a non-covalent complex formationDNA hybridization. A theoretical analysis shows that the acceleration is correlated to the catalysts surface charge density in both experimental systems and enables predicting and controlling reaction rates of like-charged compounds with counter-charged species.
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331. Peptide-based coacervate-core vesicles with semipermeable membranes
Coacervates droplets have long been considered as potential protocells to mimic living cells. However, these droplets lack a membrane and are prone to coalescence, limiting their ability to survive, interact, and organize into higher-order assemblies. This work shows that tyrosine-rich peptide conjugates can undergo liquidliquid phase separation in a well-defined pH window and transform into stable membrane-enclosed protocells by enzymatic oxidation and cross-linking at the liquidliquid interface. The oxidation of the tyrosine-rich peptides into dityrosine creates a semipermeable, flexible membrane around the coacervates with tunable thickness, which displays strong intrinsic fluorescence, and stabilizes the coacervate protocells against coalescence. The membranes have an effective molecular weight cut-off of 2.5 kDa, as determined from the partitioning of small dyes and labeled peptides, RNA, and polymers into the membrane-enclosed coacervate protocells. Flicker spectroscopy reveals a membrane bending rigidity of only 0.1kBT, which is substantially lower than phospholipid bilayers despite a larger membrane thickness. Finally, it is shown that enzymes can be stably encapsulated inside the protocells and be supplied with substrates from outside, which opens the way for these membrane-bound compartments to be used as molecularly crowded artificial cells capable of communication or as a vehicle for drug delivery.
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330. Endocytosis of coacervates into liposomes
Recent studies have shown that the interactions between condensates and biological membranes are of functional importance. Here, we study how the interaction between complex coacervates and liposomes as model systems can lead to wetting, membrane deformation, and endocytosis. Depending on the interaction strength between coacervates and liposomes, the wetting behavior ranged from nonwetting to engulfment (endocytosis) and complete wetting. Endocytosis of coacervates was found to be a general phenomenon: coacervates made from a wide range of components could be taken up by liposomes. A simple theory taking into account surface energies and coacervate sizes can explain the observed morphologies. Our findings can help to better understand condensatemembrane interactions in cellular systems and provide new avenues for intracellular delivery using coacervates.
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329. A microfluidic optimal experimental design platform for forward design of cell-free genetic networks
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.
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328. Environmental conditions drive self-organization of reaction pathways in a prebiotic reaction network
The evolution of life from the prebiotic environment required a gradual process of chemical evolution towards greater molecular complexity. Elaborate prebiotically relevant synthetic routes to the building blocks of life have been established. However, it is still unclear how functional chemical systems evolved with direction using only the interaction between inherent molecular chemical reactivity and the abiotic environment. Here we demonstrate how complex systems of chemical reactions exhibit well-defined self-organization in response to varying environmental conditions. This self-organization allows the compositional complexity of the reaction products to be controlled as a function of factors such as feedstock and catalyst availability. We observe how Breslows cycle contributes to the reaction composition by feeding C2 building blocks into the network, alongside reaction pathways dominated by formaldehyde-driven chain growth. The emergence of organized systems of chemical reactions in response to changes in the environment offers a potential mechanism for a chemical evolution process that bridges the gap between prebiotic chemical building blocks and the origin of life.
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327. A bayesian approach to extracting kinetic information from artificial enzymatic networks
In order to create artificial enzymatic networks capable of increasingly complex behavior, an improved methodology in understanding and controlling the kinetics of these networks is needed. Here, we introduce a Bayesian analysis method allowing for the accurate inference of enzyme kinetic parameters and determination of most likely reaction mechanisms, by combining data from different experiments and network topologies in a single probabilistic analysis framework. This Bayesian approach explicitly allows us to continuously improve our parameter estimates and behavior predictions by iteratively adding new data to our models, while automatically taking into account uncertainties introduced by the experimental setups or the chemical processes in general. We demonstrate the potential of this approach by characterizing systems of enzymes compartmentalized in beads inside flow reactors. The methods we introduce here provide a new approach to the design of increasingly complex artificial enzymatic networks, making the design of such networks more efficient, and robust against the accumulation of experimental errors.
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326. DNA input classification by a riboregulator-based cell-free Perceptron
The ability to recognize molecular patterns is essential for the continued survival of biological organisms, allowing them to sense and respond to their immediate environment. The design of synthetic gene-based classifiers has been explored previously; however, prior strategies have focused primarily on DNA strand-displacement reactions. Here, we present a synthetic in vitro transcription and translation (TXTL)-based perceptron consisting of a weighted sum operation (WSO) coupled to a downstream thresholding function. We demonstrate the application of toehold switch riboregulators to construct a TXTL-based WSO circuit that converts DNA inputs into a GFP output, the concentration of which correlates to the input pattern and the corresponding weights. We exploit the modular nature of the WSO circuit by changing the output protein to the Escherichia coli ?28-factor, facilitating the coupling of the WSO output to a downstream reporter network. The subsequent introduction of a ?28 inhibitor enabled thresholding of the WSO output such that the expression of the downstream reporter protein occurs only when the produced ?28 exceeds this threshold. In this manner, we demonstrate a genetically implemented perceptron capable of binary classification, i.e., the expression of a single output protein only when the desired minimum number of inputs is exceeded.
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325. Membrane reactors accelerate polymer photosensitizer-based singlet oxygen reactions
Polymer photosensitizers have gained a growing interest as singlet oxygen (1O2) generators. Although they provide a route to sustainable chemistry, their efficiency in bulk reactions is restricted by a limited oxygen supply and a short lifetime. Herein, we report membrane reactors that support a range of organic reactions using 1O2 generated from polymer photosensitizers. The porous nature of the membrane support, a nylon fabric membrane functionalized with poly(fluorenthiophen thiadiazothiophen) (PFOTTzT), allows highly efficient trapping of a thin membrane of reagents that is in close proximity to the 1O2 source. We demonstrate that oxidation reactions are accelerated by 10 100 folds, and polymerization reactions are rapidly initiated and accelerated and reach a nearly 2-fold increase in the degree of polymerization. The method is compatible with a range of solvents upon protection of the polymeric photosensitizers with a porous layer of polytetrafluoroethylene (PTFE) fabric that prevents solvent transport but allows 1O2 diffusion through. Summarizing, our membrane reactors are sustainable, low-cost, robust-in-use and overcome current limitations in reactions involving 1O2 from polymer photosensitizers and show strong potential for green industrial use.
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324. Photoswitchable molecular communication between programmable dna-based artificial membraneless organelles
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.
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323. The Effect of Geometry and TGF-? Signaling on Tumor Cell Migration from Free-Standing Microtissues
Recapitulation of 3D multicellular tissues in vitro is of great interest to the field of tumor biology to study the integrated effect of local biochemical and biophysical signals on tumor cell migration and invasion. However, most microengineered tissues and spheroids are unable to recapitulate in vitro the complexities of 3D geometries found in vivo. Here, lithographically defined degradable alginate microniches are presented to produce free-standing tumor microtissues, with precisely controlled geometry, high viability, and allowing for high cell proliferation. The role of microtissue geometry and TGF-? signaling in tumor cell migration is further investigated. TGF-? is found to induce the expression of p-myosin II, vimentin, and YAP/TAZ nuclear localization at the periphery of the microtissue, where enhanced nuclear stiffness and orientation are also observed. Upon embedding in a collagen matrix, microtissues treated with TGF-? maintain their geometric integrity, possibly due to the higher cell tension observed around the periphery. In contrast, cells in microtissues not treated with TGF-? are highly mobile and invade the surrounding matrix rapidly, with the initial migration strongly dependent on the local geometry. The microtissues presented here are promising model systems for studying the influence of biophysical properties and soluble factors on tumor cell migration.
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322. Traditional protocols and optimization methods lead to absent expression in a mycoplasma cell-free gene expression platform
Cell-free expression (CFE) systems are one of the main platforms for building synthetic cells. A major drawback is the orthogonality of cell-free systems across species. To generate a CFE system compatible with recently established minimal cell constructs, we attempted to optimize a Mycoplasma bacterium-based CFE system using lysates of the genome-minimized cell JCVI-syn3A (Syn3A) and its close phylogenetic relative Mycoplasma capricolum (Mcap). To produce mycoplasma-derived crude lysates, we systematically tested methods commonly used for bacteria, based on the S30 protocol of Escherichia coli. Unexpectedly, after numerous attempts to optimize lysate production methods or composition of feeding buffer, none of the Mcap or Syn3A lysates supported cell-free gene expression. Only modest levels of in vitro transcription of RNA aptamers were observed. While our experimental systems were intended to perform transcription and translation, our assays focused on RNA. Further investigations identified persistently high ribonuclease (RNase) activity in all lysates, despite removal of recognizable nucleases from the respective genomes and attempts to inhibit nuclease activities in assorted CFE preparations. An alternative method using digitonin to permeabilize the mycoplasma cell membrane produced a lysate with diminished RNase activity yet still was unable to support cell-free gene expression. We found that intact mycoplasma cells poisoned E. coli cell-free extracts by degrading ribosomal RNAs, indicating that the mycoplasma cells, even the minimal cell, have a surface-associated RNase activity. However, it is not clear which gene encodes the RNase. This work summarizes attempts to produce mycoplasma-based CFE and serves as a cautionary tale for researchers entering this field.
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321. A short peptide synthon for liquid–liquid phase separation
Liquid-liquid phase separation of disordered proteins has emerged as a
ubiquitous route to membraneless compartments in living cells, and
similar coacervates may have played a role when the first cells formed.
However, existing coacervates are typically made of multiple
macromolecular components, and designing short peptide analogues capable
of self-coacervation has proven difficult. Here we present a short
peptide synthon for phase separation, made of only two dipeptide
stickers linked via a flexible, hydrophilic spacer. These small-molecule
compounds self-coacervate into micrometre-sized liquid droplets at
sub-millimolar concentrations, which retain up to 75 wt% water. The
design is general and we derive guidelines for the required sticker
hydrophobicity and spacer polarity. To illustrate their potential as
protocells, we create a disulfide-linked derivative that undergoes
reversible compartmentalization controlled by redox chemistry. The
resulting coacervates sequester and melt nucleic acids, and act as
microreactors that catalyse two different anabolic reactions yielding
molecules of increasing complexity. This provides a stepping stone for
new coacervate-based protocells made of single peptide species. -
320. Single-cell intracellular epitope and transcript detection reveals signal transduction dynamics
To further our understanding of how biochemical information flows through cells upon external stimulation, we require single-cell multi-omics methods that concurrently map changes in (phospho)protein levels across signaling networks and the associated gene expression profiles. Here, we present quantification of RNA and intracellular epitopes by sequencing (QuRIE-seq), a droplet-based platform for single-cell RNA and intra- and extracellular (phospho)protein quantification through sequencing. We applied QuRIE-seq to quantify cell-state changes at both the signaling and the transcriptome level after 2-, 4-, 6-, 60-, and 180-min stimulation of the B cell receptor pathway in Burkitt lymphoma cells. Using the multi-omics factor analysis (MOFA+) framework, we delineated changes in single-cell (phospho)protein and gene expression patterns over multiple timescales and revealed the effect of an inhibitory drug (ibrutinib) on signaling and gene expression landscapes.
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319. A physicochemical orthophosphate cycle via a kinetically stable thermodynamically activated intermediate enables mild prebiotic phosphorylations
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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318. Cell-Free Characterization of Coherent Feed-Forward Loop-Based Synthetic Genetic Circuits
Regulatory pathways inside living cells employ feed-forward architectures to fulfill essential signal processing functions that aid in the interpretation of various types of inputs through noise-filtering, fold-change detection and adaptation. Although it has been demonstrated computationally that a coherent feed-forward loop (CFFL) can function as noise filter, a property essential to decoding complex temporal signals, this motif has not been extensively characterized experimentally or integrated into larger networks. Here we use post-transcriptional regulation to implement and characterize a synthetic CFFL in an Escherichia coli cell-free transcription-translation system and build larger composite feed-forward
architectures. -
317. The Dynamics of an Oscillating Enzymatic Reaction Network is Crucially Determined by Side Reactions
Synthetic complex chemical systems are often subject to perturbations in reaction conditions. To ensure robust functioning of these systems in real‐world applications, a better understanding is required of how resilience to perturbations could be included in the design of these systems. In order to develop such an understanding we need a deeper insight into how chemical systems respond to perturbations. Here, we study the effect of spiking concentrations in an oscillating enzymatic reaction network. We identify that a different magnitude of a perturbation triggers two distinctive responses: obtaining sustained oscillations and causing the loss of the amplitude. We rationalise our findings based on non‐linear dynamics and identify that non‐essential
side reactions crucially tailor the observed behaviour. -
316. Reversible Photoswitchable Inhibitors Enable Wavelength-Selective Regulation of Out-of-Equilibrium Bi-enzymatic Systems
The construction of synthetic enzymatic reaction networks can provide new insights into the design principles of living systems. However, the programmable connection of enzymes into a wide range of network topologies has been challenging due to the lack of a general strategy enabling a reversible activity regulation of individual network enzymes. Here, we exploit a general and modular strategy based on the external regulation of enzymes using light and photoswitchable inhibitors (PIs) that enables the bottom-up construction and control of enzymatic systems studied under out-of-equilibrium conditions. Upon synthesis and incorporation of potent photoswitchable trypsin inhibitors (Tr-PIs), the output of several functional enzymatic systems could be photoregulated using 390/460 nm light as a trigger signal. In addition, the wavelength-selective control over the activity of two enzymes within a functional bi-enzymatic system was achieved using a suitable combination of two PIs.
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315. Reversible Photoswitchable Inhibitors Generate Ultrasensitivity in Out-of-Equilibrium Enzymatic Reactions
Ultrasensitivity is a ubiquitous emergent property of biochemical
reaction networks. The design and construction of synthetic reaction
networks exhibiting ultrasensitivity has been challenging, but would
greatly expand the potential properties of life-like materials. Herein,
we exploit a general and modular strategy to reversibly regulate the
activity of enzymes using light and show how ultrasensitivity arises in
simple out-of-equilibrium enzymatic systems upon incorporation of
reversible photoswitchable inhibitors (PIs). Utilizing a
chromophore/warhead strategy, PIs of the protease α-chymotrypsin were
synthesized, which led to the discovery of inhibitors with large
differences in inhibition constants (Ki) for the
different photoisomers. A microfluidic flow setup was used to study
enzymatic reactions under out-of-equilibrium conditions by continuous
addition and removal of reagents. Upon irradiation of the continuously
stirred tank reactor with different light pulse sequences, i.e., varying
the pulse duration or frequency of UV and blue light irradiation,
reversible switching between photoisomers resulted in ultrasensitive
responses in enzymatic activity as well as frequency filtering of input
signals. This general and modular strategy enables reversible and
tunable control over the kinetic rates of individual enzyme-catalyzed
reactions and makes a programmable linkage of enzymes to a wide range of
network topologies feasible. -
314. One-Step Generation of Multisomes from Lipid-Stabilized Double Emulsions
Multisomes are multicompartmental structures formed by a lipid-stabilized network of aqueous droplets, which are contained by an outer oil phase. These biomimetic structures are emerging as a versatile platform for soft matter and synthetic biology applications. While several methods for producing multisomes have been described, including microfluidic techniques, approaches for generating biocompatible, monodisperse multisomes in a reproducible manner remain challenging to implement due to low throughput and complex device fabrication. Here, we report on a robust method for the dynamically controlled generation of multisomes with controllable sizes and high monodispersity from lipid-based double emulsions. The described microfluidic approach entails the use of three different phases forming a water/oil/water (W/O/W) double emulsion stabilized by lipid layers. We employ a gradient of glycerol concentration between the inner core and outer phase to drive the directed osmosis, allowing the swelling of lamellar lipid layers resulting in the formation of small aqueous daughter droplets at the interface of the inner aqueous core. By adding increasing concentrations of glycerol to the outer aqueous phase and subsequently varying the osmotic gradient, we show that key structural parameters, including the size of the internal droplets, can be specifically controlled. Finally, we show that this approach can be used to generate multisomes encapsulating small-molecule cargo, with potential applications in synthetic biology, drug delivery, and as carriers for active materials in the food and cosmetics industries.
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313. Energy expenditure during cell spreading influences the cellular response to matrix stiffness
Cells respond to the mechanical properties of the extracellular matrix (ECM) through formation of focal adhesions (FAs), re-organization of the actin cytoskeleton and adjustment of cell contractility. These are energy demanding processes, but a potential causality between mechanical cues (matrix stiffness) and cellular (energy) metabolism remains largely unexplored. Here, we cultured human mesenchymal stem cells (hMSCs) on stiff (20 kPa) or soft (1 kPa) substrate and demonstrate that cytoskeletal reorganization and FA formation spreading on stiff substrates lead to a drop in intracellular ATP levels, correlating with activation of AMP-activated protein kinase (AMPK). The resulting increase in ATP levels further facilitates cell spreading and reinforces cell tension of the steady state, and coincides with nuclear localization of YAP/TAZ and Runx2. While on soft substrates (1 kPa), lowered ATP levels limit these cellular mechanoresponses. Furthermore, genetic ablation of AMPK lowered cellular ATP levels on stiff substrate and strongly reduced responses to substrate stiffness. Together, these findings reveal a hitherto unidentified relationship between energy expenditure and the cellular mechanoresponse, and point to AMPK as a key mediator of stem cell fate in response to ECM mechanics.
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312. Intelligent Microfluidics: The Convergence of Machine Learning and Microfluidics in Materials Science and Biomedicine
Microfluidics permit the automated manipulation of fluids at the microscale with high throughput and spatiotemporal precision, enabling the generation of large, multidimensional datasets. Machine intelligence provides powerful predictive tools with the ability to learn from data. The analysis of microfluidics-generated data via machine learning has been applied in a variety of contexts, achieving impressive results. Here, we elaborate on the potential of operating microfluidic platforms via closed-loop data-driven models by leveraging multimodal monitoring and data-acquisition instrumentation. We believe this approach will provide a robust framework for fundamental explorations in materials science and biomedicine, with implications in fields such as drug discovery, nanomaterials, in vitro organ modeling, and developmental biology. We identify challenges and propose research strategies in the context of the prediction and optimization of chemical reactions and materials syntheses and the development of the next generation of more robust and functional organs-on-chips and emerging organoids-on-chips.
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311. Transcription and Translation in Cytomimetic Protocells Perform Most Efficiently at Distinct Macromolecular Crowding Conditions
The formation of rytomimetic protocells that capture the physicochemical aspects of living cells is an important goal in bottom-up synthetic biology. Here, we recreated the crowded cytoplasm in liposome-based protocells and studied the kinetics of cell-free gene expression in these crowded containers. We found that diffusion of key components is affected not only by macromolecular crowding but also by enzymatic activity in the protocell. Surprisingly, size-dependent diffusion in crowded conditions yielded two distinct maxima for protein synthesis, reflecting the differential impact of crowding on transcription and translation. Our experimental data show, for the first time, that macromolecular crowding induces a switch from reaction to diffusion control and that this switch depends on the sizes of the macromolecules involved. These results highlight the need to control the physical environment in the design of synthetic cells.
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310. Autonomous mesoscale positioning emerging from myelin filament self-organization and Marangoni flows
Out-of-equilibrium molecular systems hold great promise as dynamic, reconfigurable matter that executes complex tasks autonomously. However, translating molecular scale dynamics into spatiotemporally controlled phenomena emerging at mesoscopic scale remains a challenge-especially if one aims at a design where the system itself maintains gradients that are required to establish spatial differentiation. Here, we demonstrate how surface tension gradients, facilitated by a linear amphiphile molecule, generate Marangoni flows that coordinate the positioning of amphiphile source and drain droplets floating at air-water interfaces. Importantly, at the same time, this amphiphile leads, via buckling instabilities in lamellar systems of said amphiphile, to the assembly of millimeter long filaments that grow from the source droplets and get absorbed at the drain droplets. Thereby, the Marangoni flows and filament organization together sustain the autonomous positioning of interconnected droplet-filament networks at the mesoscale. Our concepts provide potential for the development of non-equilibrium matter with spatiotemporal programmability. Buckling instabilities in amphiphile-based lamellar systems can lead to the formation of tubular fingers. Van der Weijden et al. show how to shepherd their growth and destination by using drain droplets that help establishing stable interconnected mesoscale droplet networks.
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309. Probing single-cell metabolism reveals prognostic value of highly metabolically active circulating stromal cells in prostate cancer
Despite their important role in metastatic disease, no general method to detect circulating stromal cells (CStCs) exists. Here, we present the Metabolic Assay-Chip (MA-Chip) as a label-free, droplet-based microfluidic approach allowing single-cell extracellular pH measurement for the detection and isolation of highly metabolically active cells (hm-cells) from the tumor microenvironment. Single-cell mRNA-sequencing analysis of the hm-cells from metastatic prostate cancer patients revealed that approximately 10% were canonical EpCAM(+) hm-CTCs, 3% were EpCAM(-) hm-CTCs with up-regulation of prostate-related genes, and 87% were hm-CStCs with profiles characteristic for cancer-associated fibroblasts, mesenchymal stem cells, and endothelial cells. Kaplan-Meier analysis shows that metastatic prostate cancer patients with more than five hm-cells have a significantly poorer survival probability than those with zero to five hm-cells. Thus, prevalence of hm-cells is a prognosticator of poor outcome in prostate cancer, and a potentially predictive and therapy response biomarker for agents cotargeting stromal components and preventing epithelial-to-mesenchymal transition.
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308. Microfabricated Gaps Reveal the Effect of Geometrical Control in Wound Healing
The geometry (size and shape) of gaps is a key determinant in controlling gap closure during wound healing. However, conventional methods for creating gaps result in un-defined geometries and poorly characterized conditions (cell death factors and cell debris), which can influence the gap closure process. To overcome these limitations, a novel method to create well-defined geometrical gaps is developed. First, smooth muscle cells (SMCs) are seeded in variously shaped micro-containers made out of hyaluronic acid hydrogels. Cell proliferation and cell tension induce fibrous collagen production by SMCs predominantly around the edges of the micro-containers. Upon removal of SMCs, the selectively deposited collagen results in micro-containers with cell-adhesive regions along the edges and walls. Fibroblasts are seeded in these micro-containers, and upon attaching and spreading, they naturally form gaps with different geometries. The rapid proliferation of fibroblasts from the edge results in filling and closure of the gaps. It is demonstrated that gap closure rate as well as closure mechanism is strongly influenced by geometrical features, which points to an important role for cellular tension and cell proliferation in gap closure.
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307. Early warning signals in chemical reaction networks
Complex systems such as ecosystems, the climate and stock markets produce emergent behaviour which is capable of undergoing dramatic change when pushed beyond a tipping point. Such complex systems display Early Warning Signals in their behaviour when they are close to a tipping point. Here we show that a complex chemical reaction network can also display early warning signals when it is in close proximity to the boundary between oscillatory and steady state concentration behaviours. We identify early warning signals using both an active sensing method, based on the recovery time of an oscillatory response after a perturbation in temperature, and a passive sensing method, based upon a change in the shape of the oscillations. The presence of the early warning signals indicates that complex, dissipative chemical networks can intrinsically sense their proximity to a boundary between behaviours.
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306. Dysmetabolic Circulating Tumor Cells Are Prognostic in Metastatic Breast Cancer
Circulating tumor cells (CTCs) belong to a heterogeneous pool of rare cells, and a unequivocal phenotypic definition of CTC is lacking. Here, we present a definition of metabolically-altered CTC (MBA-CTCs) as CD45-negative cells with an increased extracellular acidification rate, detected with a single-cell droplet microfluidic technique. We tested the prognostic value of MBA-CTCs in 31 metastatic breast cancer patients before starting a new systemic therapy (T0) and 3-4 weeks after (T1), comparing results with a parallel FDA-approved CellSearch (CS) approach. An increased level of MBA-CTCs was associated with: i) a shorter median PFS pre-therapy (123 days vs. 306; p < 0.0001) and during therapy (139 vs. 266 days; p = 0.0009); ii) a worse OS pre-therapy (p = 0.0003, 82% survival vs. 20%) and during therapy (p = 0.0301, 67% survival vs. 38%); iii) good agreement with therapy response (kappa = 0.685). The trend of MBA-CTCs over time (combining data at T0 and T1) added information with respect to separate evaluation of T0 and T1. The combined results of the two assays (MBA and CS) increased stratification accuracy, while correlation between MBA and CS was not significant, suggesting that the two assays are detecting different CTC subsets. In conclusion, this study suggests that MBA allows detection of both EpCAM-negative and EpCAM-positive, viable and label-free CTCs, which provide clinical information apparently equivalent and complementary to CS. A further validation of proposed method and cut-offs is needed in a larger, separate study.
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305. Dynamic Environments as a Tool to Preserve Desired Output in a Chemical Reaction Network
Current efforts to design functional molecular systems have overlooked the importance of coupling out-of-equilibrium behaviour with changes in the environment. Here, the authors use an oscillating reaction network and demonstrate that the application of environmental forcing, in the form of periodic changes in temperature and in the inflow of the concentration of one of the network components, removes the dependency of the periodicity of this network on temperature or flow rates and enforces a stable periodicity across a wide range of conditions. Coupling a system to a dynamic environment can thus be used as a simple tool to regulate the output of a network. In addition, the authors show that coupling can also induce an increase in behavioural complexity to include quasi-periodic oscillations.
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304. Unravelling Receptor and RGD Motif Dependence of Retargeted Adenoviral Vectors using Advanced Tumor Model Systems
Recent advances in engineering adenoviruses are paving the way for new therapeutic gene delivery approaches in cancer. However, there is limited knowledge regarding the impact of adenoviral retargeting on transduction efficiency in more complex tumor architectures, and the role of the RGD loop at the penton base in retargeting is unclear. To address this gap, we used tumor models of increasing complexity to study the role of the receptor and the RGD motif. Employing tumor-fibroblast co-culture models, we demonstrate the importance of the RGD motif for efficient transduction in 2D through the epithelial cell adhesion molecule (EpCAM), but not the epidermal growth factor receptor (EGFR). Via optical clearing of co-culture spheroids, we show that the RGD motif is required for transduction via both receptors in 3D tumor architectures. We subsequently employed a custom-designed microfluidic model containing collagen-embedded tumor spheroids, mimicking the interplay between interstitial flow, extracellular matrix and adenoviral transduction. Image analysis of on-chip cleared spheroids indicated the importance of the RGD motif for on-chip adenoviral transduction. Together, our results show the interrelationship between receptor characteristics, the RGD motif, the 3D tumor architecture and retargeted adenoviral transduction efficiency. The findings are important for the rational design of next-generation therapeutic adenoviruses.
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303. Single-Cell Analysis Using Droplet Microfluidics
Droplet microfluidics has revolutionized the study of single cells. The ability to compartmentalize cells within picoliter droplets in microfluidic devices has opened up a wide range of strategies to extract information at the genomic, transcriptomic, proteomic, or metabolomic level from large numbers of individual cells. Studying the different molecular landscapes at single-cell resolution has provided the authors with a detailed picture of intracellular heterogeneity and the resulting changes in cellular phenotypes. In addition, these technologies have aided in the discovery of rare cells in tumors or in the immune system, and left the authors with a deeper understanding of the fundamental biological processes that determine cell fate. This review aims to provide a detailed overview of the various droplet microfluidic strategies reported in the literature, taking into account the sometimes subtle differences in workflow or reagents that enable or improve certain protocols. Specifically, approaches to targeted- and whole-genome analysis, as well as whole-transcriptome profiling techniques, are reviewed. In addition, an up-to-date overview of new methods to characterize and quantify single-cell protein levels, and of developments to screen secreted molecules such as antibodies, cytokines, or metabolites at the single-cell level, is provided.
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302. On the importance of reaction networks for synthetic living systems
The goal of creating a synthetic cell necessitates the development of reaction networks which will underlie all of its behaviours. Recent developments in in vitro systems, based upon both DNA and enzymes, have created networks capable of a range of behaviours e.g. information processing, adaptation and diffusive signalling. These networks are based upon reaction motifs that when combined together produce more complex behaviour. We highlight why it is inevitable that networks, based on enzymes or enzyme-like catalysts, will be required for the construction of a synthetic cell. We outline several of the challenges, including (a) timing, (b) regulation and (c) energy distribution, that must be overcome in order to transition from the simple networks we have today to much more complex networks capable of a variety of behaviours and which could find application one day within a synthetic cell.
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301. Branched DNA Architectures Produced by PCR-Based Assembly as Gene Compartments for Cell-Free Gene-Expression Reactions
The physical distance between genes plays important roles in controlling gene expression reactions in vivo. Herein, we report the design and synthesis of a branched gene architecture in which three transcription units are integrated into one framework through assembly based on the polymerase chain reaction (PCR), together with the exploitation of these constructs as gene compartments for cell-free gene expression reactions, probing the impact of this physical environment on gene transcription and translation. We find that the branched gene system enhances gene expression yields, in particular at low concentrations of DNA and RNA polymerase (RNAP); furthermore, in a crowded microenvironment that mimics the intracellular microenvironment, gene expression from branched genes maintains a relatively high level. We propose that the branched gene assembly forms a membrane-free gene compartment that resembles the nucleoid of prokaryotes and enables RNAP to shuttle more efficiently between neighboring transcription units, thus enhancing gene expression efficiency. Our branched DNA architecture provides a valuable platform for studying the influence of cellular physical environments on biochemical reactions in simplified cell-free systems.
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300. Modular Design of Small Enzymatic Reaction Networks Based on Reversible and Cleavable Inhibitors
Systems chemistry aims to mimic the functional behavior of living systems by constructing chemical reaction networks with well-defined dynamic properties. Enzymes can play a key role in such networks, but there is currently no general and scalable route to the design and construction of enzymatic reaction networks. Here, we introduce reversible, cleavable peptide inhibitors that can link proteolytic enzymatic activity into simple network motifs. As a proof-of-principle, we show auto-activation topologies producing sigmoidal responses in enzymatic activity, explore cross-talk in minimal systems, design a simple enzymatic cascade, and introduce non-inhibiting phosphorylated peptides that can be activated using a phosphatase.
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299. A Multilayer Microfluidic Platform for the Conduction of Prolonged Cell-Free Gene Expression
The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic regulatory circuits. The analysis of synthetic genetic regulatory networks in vivo is time consuming and suffers from a lack of environmental control, with exogenous synthetic components interacting with host processes resulting in undesired behavior. To overcome these issues, cell-free characterization of novel circuitry is becoming more prevalent. In vitro transcription and translation (IVTT) mixtures allow the regulation of the experimental environment and can be optimized for each unique system. The protocols presented here detail the fabrication of a multilayer microfluidic device that can be utilized to sustain IVTT reactions for prolonged durations. In contrast to batch reactions, where resources are depleted over time and (by-) products accumulate, the use of microfluidic devices allows the replenishment of resources as well as the removal of reaction products. In this manner, the cellular environment is emulated by maintaining an out-of-equilibrium environment in which the dynamic behavior of gene circuits can be investigated over extended periods of time. To fully exploit the multilayer microfluidic device, hardware and software have been integrated to automate the IVTT reactions. By combining IVTT reactions with the microfluidic platform presented here, it becomes possible to comprehensively analyze complex network behaviors, furthering our understanding of the mechanisms that regulate cellular processes.
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298. Cell-free microcompartmentalised transcription-translation for the prototyping of synthetic communication networks
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.
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297. Robustness, Entrainment, and Hybridization in Dissipative Molecular Networks, and the Origin of Life
How simple chemical reactions self-assembled into complex, robust networks at the origin of life is unknown. This general problem-self-assembly of dissipative molecular networks-is also important in understanding the growth of complexity from simplicity in molecular and biomolecular systems. Here, we describe how heterogeneity in the composition of a small network of oscillatory organic reactions can sustain (rather than stop) these oscillations, when homogeneity in their composition does not. Specifically, multiple reactants in an amide-forming network sustain oscillation when the environment (here, the space velocity) changes, while homogeneous networks-those with fewer reactants-do not. Remarkably, a mixture of two reactants of different structure-neither of which produces oscillations individually-oscillates when combined. These results demonstrate that molecular heterogeneity present in mixtures of reactants can promote rather than suppress complex behaviors.
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296. Catalytic transport of molecular cargo using diffusive binding along a polymer track
Transport at the molecular scale is a prerequisite for the development of future molecular factories. Here, we have designed oligoanionic molecular sliders on polycationic tracks that exploit Brownian motion and diffusive binding to transport cargo without using a chemical fuel. The presence of the polymer tracks increases the rate of bimolecular reactions between modified sliders by over two orders of magnitude. Molecular dynamics simulations showed that the sliders not only diffuse, but also jump and hop surprisingly efficiently along polymer tracks. Inspired by acetyl-coenzyme A transporting and delivering acetyl groups in many essential biochemical processes, we developed a new and unconventional type of catalytic transport involving sliders (including coenzyme A) picking up, transporting and selectively delivering molecular cargo. Furthermore, we show that the concept of diffusive binding can also be utilized for the spatially controlled transport of chemical groups across gels. This work represents a new concept for designing functional nanosystems based on random Brownian motion.
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295. Combined quantification of intracellular (phospho-)proteins and transcriptomics from fixed single cells
Environmental stimuli often lead to heterogeneous cellular responses and transcriptional output. We developed single-cell RNA and Immunodetection (RAID) to allow combined analysis of the transcriptome and intracellular (phospho-)proteins from fixed single cells. RAID successfully recapitulated differentiation-state changes at the protein and mRNA level in human keratinocytes. Furthermore, we show that differentiated keratinocytes that retain high phosphorylated FAK levels, a feature associated with stem cells, also express a selection of stem cell associated transcripts. Our data demonstrates that RAID allows investigation of heterogeneous cellular responses to environmental signals at the mRNA and phospho-proteome level.
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294. Cellular Volume and Matrix Stiffness Direct Stem Cell Behavior in a 3D Microniche
The central question addressed in this study is whether cells with different sizes have different responses to matrix stiffness. We used methacrylated hyaluronic acid (MeHA) hydrogels as the matrix to prepare an in vitro 3D microniche in which the single stem cell volume and matrix stiffness can be altered independently from each other. This simple approach enabled us to decouple the effects of matrix stiffness and cell volume in 3D microenvironments. Human mesenchymal stem cells (hMSCs) were cultured in individual 3D microniches with different volumes (2800, 3600, and 6000 mu m(3)) and stiffnesses (5, 12, and 23 kPa). We demonstrated that cell volume affected the cellular response to matrix stiffness. When cells had an optimal volume, cells could form clear stress fibers and focal adhesions on soft, intermediate, or stiff matrix. In small cells, stress fiber formation and YAP/TAZ localization were not affected by stiffness. This study highlights the importance of considering cellular volume and substrate stiffness as important cues governing cell-matrix interactions.
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293. Sigma Factor-Mediated Tuning of Bacterial Cell-Free Synthetic Genetic Oscillators
Cell-free transcription-translation provides a simplified prototyping environment to rapidly design and study synthetic networks. Despite the presence of a well characterized toolbox of genetic elements, examples of genetic networks that exhibit complex temporal behavior are scarce. Here, we present a genetic oscillator implemented in an E. coli-based cell-free system under steady-state conditions using microfluidic flow reactors. The oscillator has an activator-repressor motif that utilizes the native transcriptional machinery of E. coli: the RNAP and its associated sigma factors. We optimized a kinetic model with experimental data using an evolutionary algorithm to quantify the key regulatory model parameters. The functional modulation of the RNAP was investigated by coupling two oscillators driven by competing sigma factors, allowing the modification of network properties by means of passive transcriptional regulation.
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292. Bottom-Up Construction of an Adaptive Enzymatic Reaction Network
The reproduction of emergent behaviors in nature using reaction networks is an important objective in synthetic biology and systems chemistry. Herein, the first experimental realization of an enzymatic reaction network capable of an adaptive response is reported. The design is based on the dual activity of trypsin, which activates chymotrypsin while at the same time generating a fluorescent output from a fluorogenic substrate. Once activated, chymotrypsin counteracts the trypsin output by competing for the fluorogenic substrate and producing a non-fluorescent output. It is demonstrated that this network produces a transient fluorescent output under out-of-equilibrium conditions while the input signal persists. Importantly, in agreement with mathematical simulations, we show that optimization of the pulse-like response is an inherent trade-off between maximum amplitude and lowest residual fluorescence.
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291. Fabrication of 3D Tubular Hydrogel Materials through On-Site Surface Free Radical Polymerization
Constructing tubular hydrogel materials with desirable structures based on their functional application is a big challenge. Here, we report a simple but effective method to prepare tubular hydrogels with complex geometries by surface radical polymerization, in which an iron wire acts as both catalyst and template for the formation of a gel layer with controllable thickness. The formed hydrogel layer can be easily peeled off from the template after secondary cross-linking to obtain hollow hydrogel tubes which exhibit extraordinary and tunable tensile strength, good elasticity, and pressure-bearing capability. The method can be generalized to construct a series of complex three-dimensional hydrogel tubes with versatile components for building up fluidic channels or biocompatible 3D cell culturing platform for tissue engineering. Such a method is a great advance in the field of hydrogel materials. It is anticipated that this innovation would open up the door for developing functional 3D tubular hydrogel materials suitable for multiple applications.
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290. Microfluidic-Assisted Fabrication of Clay Microgels for Cell-Free Protein Synthesis
Cell-free protein synthesis (CFPS) is a robust platform for the simple, rapid, and cost-effective in vitro production of proteins, as well as an important tool for cell-free synthetic biology research. Here, a microfluidic clay microgel system is reported, which creates compartmentalized microenvironments for CFPS capable of high-yield and repeated protein synthesis, as well as an artificial cell-like structure. As an advantageous platform for CFPS, a modular manner to prepare clay microgels with rationally designed functions is demonstrated: (i) gene/clay microgels enhance protein expression, (ii) gene/clay/magnetic nanoparticle microgels enable a repeated protein production system, and (iii) gene/clay microgels in microfluidic droplets serve as a cell-like structure. Beyond CFPS, considering the compatibility of clay microgels with hydrophilic functional materials, our clay microgels will provide a more general platform for preparing a variety of functional materials such as encapsulating drugs and cells, enabling more biomedical applications.
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289. Dissipative adaptation in driven self-assembly leading to self-dividing fibrils
Out-of-equilibrium self-assembly of proteins such as actin and tubulin is a key regulatory process controlling cell shape, motion and division. The design of functional nanosystems based on dissipative self-assembly has proven to be remarkably difficult due to a complete lack of control over the spatial and temporal characteristics of the assembly process. Here, we show the dissipative self-assembly of FtsZ protein (a bacterial homologue of tubulin) within coacervate droplets. More specifically, we show how such barrier-free compartments govern the local availability of the energy-rich building block guanosine triphosphate, yielding highly dynamic fibrils. The increased flux of FtsZ monomers at the tips of the fibrils results in localized FtsZ assembly, elongation of the coacervate compartments, followed by division of the fibrils into two. We rationalize the directional growth and division of the fibrils using dissipative reaction-diffusion kinetics and capillary action of the filaments as main inputs. The principle presented here, in which open compartments are used to modulate the rates of dissipative self-assembly by restricting the absorption of energy from the environment, may provide a general route to dissipatively adapting nanosystems exhibiting life-like behaviour.
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288. Single-cell analysis reveals that stochasticity and paracrine signaling control interferon-alpha production by plasmacytoid dendritic cells
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 TNF alpha production is limited to a small sub-population 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 micro-environment, 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.
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287. Recent Advances in Engineering the Stem Cell Microniche in 3D
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.
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286. Macromolecularly Crowded Protocells from Reversibly Shrinking Monodisperse Liposomes
The compartmentalization of cell-free gene expression systems in liposomes provides an attractive route to the formation of protocells, but these models do not capture the physical (crowded) environment found in living systems. Here, we present a microfluidics-based route to produce monodisperse liposomes that can shrink almost 3 orders of magnitude without compromising their stability. We demonstrate that our strategy is compatible with cell-free gene expression and show increased protein production rates in crowded liposome protocells.
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285. 3D microniches reveal the importance of cell size and shape
Geometrical cues have been shown to alter gene expression and differentiation on 2D substrates. However, little is known about how geometrical cues affect cell function in 3D. One major reason for this lack of understanding is rooted in the difficulties of controlling cell geometry in a complex 3D setting and for long periods of culture. Here, we present a robust method to control cell volume and shape of individual human mesenchymal stem cells (hMSCs) inside 3D microniches with a range of different geometries (e.g., cylinder, triangular prism, cubic, and cuboid). We find that the actin filaments, focal adhesions, nuclear shape, YAP/TAZ localization, cell contractility, nuclear accumulation of histone deacetylase 3, and lineage selection are all sensitive to cell volume. Our 3D microniches enable fundamental studies on the impact of biophysical cues on cell fate, and have potential applications in investigating how multicellular architectures organize within geometrically well-defined 3D spaces.
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284. Rational design and dynamics of self-propelled colloidal bead chains: from rotators to flagella
The quest for designing new self-propelled colloids is fuelled by the demand for simple experimental models to study the collective behaviour of their more complex natural counterparts. Most synthetic self-propelled particles move by converting the input energy into translational motion. In this work we address the question if simple self-propelled spheres can assemble into more complex structures that exhibit rotational motion, possibly coupled with translational motion as in flagella. We exploit a combination of induced dipolar interactions and a bonding step to create permanent linear bead chains, composed of self-propelled Janus spheres, with a well-controlled internal structure. Next, we study how flexibility between individual swimmers in a chain can affect its swimming behaviour. Permanent rigid chains showed only active rotational or spinning motion, whereas longer semi-flexible chains showed both translational and rotational motion resembling flagella like-motion, in the presence of the fuel. Moreover, we are able to reproduce our experimental results using numerical calculations with a minimal model, which includes full hydrodynamic interactions with the fluid. Our method is general and opens a new way to design novel self-propelled colloids with complex swimming behaviours, using different complex starting building blocks in combination with the flexibility between them.
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283. Photochemical Control over Oscillations in Chemical Reaction Networks
Systems chemistry aims to emulate the functional behavior observed in living systems by constructing chemical reaction networks (CRNs) with well-defined dynamic properties. Future expansion of the complexity of these systems would require external control to tune behavior and temporal organization of such CRNs. In this work, we design and implement a photolabile probe, which upon irradiation strengthens the negative feedback loop of a CRN that produces oscillations of trypsin under out-of-equilibrium conditions. By changing the timing and duration of irradiation, we can tailor the temporal response of the network.
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282. Adaptation trajectories during adhesion and spreading affect future cell states
Cells are complex systems in which dynamic gene expression and protein-interaction networks adapt to changes in the environment. Seeding and subsequent spreading of cells on substrates represents an example of adaptation to a major perturbation. The formation of adhesive interactions and self-organisation of the cytoskeleton during initial spreading might prime future cell behaviour. To elucidate the role of these events on later cellular behaviour, we mapped the trajectories by which cells respond to seeding on substrates with different physical properties. Our experiments on cell spreading dynamics on collagen-or fibrin-coated polyacrylamide gels and collagen or fibrin hydrogels show that on each substrate, cells follow distinct trajectories of morphological changes, culminating in fundamentally different cell states as quantified by RNA-expression levels, YAP/TAZ localisation, proliferation and differentiation propensities. The continuous adaptation of the cell to environmental cues leaves traces due to differential cellular organisation and gene expression profiles, blurring correlations between a particular physical property and cellular phenotype.
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281. A trypsin-based bistable switch
Recreating some of the emergent behavior seen in biological reaction networks is an important goal in the new field of systems chemistry. One of the classic examples of complex behavior is bistability, which is abundantly used in living organisms for switching between cellular states. Here, we create a bistable switch based on the autocatalytic activation and inhibition of the enzyme trypsin under flow conditions. We investigate the influence of the inhibitor structure, and hence inhibition kinetics, on the properties of the bistable switch. (C) 2017 Elsevier Ltd. All rights reserved.
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280. Microfluidic Formation of Monodisperse Coacervate Organelles in Liposomes
Coacervates have been widely studied as model compartments in protocell research. Complex coacervates composed of disordered proteins and RNA have also been shown to play an important role in cellular processes. Herein, we report on a microfluidic strategy for constructing monodisperse coacervate droplets encapsulated within uniform unilamellar liposomes. These structures represent a bottomup approach to hierarchically structured protocells, as demonstrated by storage and release of DNA from the encapsulated coacervates as well as localized transcription.
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279. Grip on complexity in chemical reaction networks
A new discipline of systems chemistry is emerging, which aims to capture the complexity observed in natural systems within a synthetic chemical framework. Living systems rely on complex networks of chemical reactions to control the concentration of molecules in space and time. Despite the enormous complexity in biological networks, it is possible to identify network motifs that lead to functional outputs such as bistability or oscillations. To truly understand how living systems function, we need a complete understanding of how chemical reaction networks (CRNs) create function. We propose the development of a bottom-up approach to design and construct CRNs where we can follow the influence of single chemical entities on the properties of the network as a whole. Ultimately, this approach should allow us to not only understand such complex networks but also to guide and control their behavior.
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278. Molecular Engineering of Robustness and Resilience in Enzymatic Reaction Networks
Living systems rely on complex networks of chemical reactions to control the concentrations of molecules in space and time. Despite the enormous complexity in biological networks, it is possible to identify network motifs that lead to functional outputs such as bistability or oscillations. One of the greatest challenges in chemistry is the creation of such functionality from chemical reactions. A key limitation is our lack of understanding of how molecular structure impacts on the dynamics of chemical reaction networks, preventing the design of networks that are robust (i.e., function in a large parameter space) and resilient (i.e., reach their out-of-equilibrium function rapidly). Here we demonstrate that reaction rates of individual reactions in the network can control the dynamics by which the system reaches limit cycle oscillations, thereby gaining information on the key parameters that govern the dynamics of these networks. We envision that these principles will be incorporated into the design of network motifs, enabling chemists to develop molecular software to create functional behavior in chemical systems.
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277. Collagen Gels with Different Fibrillar Microarchitectures Elicit Different Cellular Responses
The extracellular matrix consists of a complex mixture of fibrillar proteins, in which the architecture and mechanical properties of the protein fibrils vary considerably in various tissues. Here, we systematically polymerized collagen gels at different temperatures, providing substrates with tunable mechanics and defined local microarchitecture. We studied the dependence of spreading dynamics, proliferation, migration, and differentiation of human mesenchymal stem cells (hMSCs) on the fibrillar properties as compared to the bulk properties of the matrix. We found that high fiber stiffness, together with shorter fiber lengths, limited the transfer of cellular traction forces to nearby fibers. As a result, cells were not able to build up sufficient tension, which suppressed cell spreading, proliferation, and migration. Cells on such fibers also showed limited focal adhesion formation and different lineage selection preferences. In contrast, cell spreading, proliferation, and migration was always associated with fiber recruitment, long-range deformations in the collagen gel networks and an increase in collagen density around cells. Typically, cells on such substrates had a preference for osteogenic differentiation and showed higher levels of focal adhesions formation. These results contribute to a further understanding of the mechanotransduction process and to the design criteria for future biomimetic materials for tissue-engineering applications.
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276. Evidence of Ion-Pairing in Cationic Brushes from Evaluation of Brush Charging and Structure by Electrokinetic and Surface Conductivity Analysis
Strong cationic poly(2-(methacryloyloxy)-ethyltrimethylammonium chloride) (PMETAC) brushes are widely employed as platform for studying fundamental physicochemical properties of permanently charged polymers at interfaces. We report here a detailed analysis of the Structure, interfacial and bulk charging of PMETAC brushes over a broad range of pH values and salt concentrations (pH 2.5-9.5, 0.01-10 mM KCl electrolyte). Streaming current and Surface conductivity measurements were quantitatively analyzed on the basis of a recently developed theory for electrohydrodynamics at diffuse soft interfaces under lateral flow conditions taking into account ion pairing at low salt concentrations. In addition, the effects of different chaotropic anions on brush. charge and thickness were deciphered from interpretation of surface conductivity data collected in 1 mM KNO3, KI, and KClO4 electrolytes. In combination, confrontation between theory and experiments reveals the existence of a PMETAC segment density gradient at the brush/solution interphase that is independent of pH and KCl concentrations above 0.1 mM. With decreasing salt content at pH 6 from 0.1 mM to 0.01 mM, the corresponding nonmonotonous variation of streaming current indicates electrostatically driven interfacial swelling of the brush, while the accompanied variation in surface conductivity reveals a dramatic decrease in the net density of structural charges Within the bulk material. The latter feature is assigned to the occurrence of pairing between chloride anions from background electrolyte and quaternary ammonium groups supported by PMETAC chains. The decrease of the net charge density in the bulk of the brush is Shown to be more pronounced with increasing anion hydrophobicity, i.e., upon promoting the ion-pairing process that subsequently leads to a shrinking of the brush volume. The study illustrates the complementary information derived from surface conductivity and streaming current analysis: the former reflects electrohydrodynamic processes hi the bulk of the brush, whereas the latter primarily mirrors professes at the polymer/solution interphase. It further demonstrates that electrokinetic and surface conductivity are valuable tools for demonstrating and monitoring ion-pairing processes useful to reversibly tune macrostopic properties such as brush swelling or stiffness.
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275. Preprogramming Complex Hydrogel Responses using Enzymatic Reaction Networks
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.
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274. Microfluidic Assembly of Monodisperse Vesosomes as Artificial Cell Models
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.
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273. Protein Synthesis in Coupled and Uncoupled Cell-Free Prokaryotic Gene Expression Systems
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.
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272. A Compartmentalized Out-of-Equilibrium Enzymatic Reaction Network for Sustained Autonomous Movement
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.
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271. A Method for Detecting Circulating Tumor Cells Based on the Measurement of Single-Cell Metabolism in Droplet-Based Microfluidics
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.
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270. The nanotechnology of life-inspired systems
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.
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269. Monodisperse Uni- and Multicompartment Liposomes
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 itm, 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.
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268. Quantitative Single-Cell mRNA Analysis in Hydrogel Beads
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.
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267. Bio-Inspired Renewable Surface-Initiated Polymerization from Permanently Embedded Initiators
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.
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266. Cell-Like Nanostructured Environments Alter Diffusion and Reaction Kinetics in Cell-Free Gene Expression
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.
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265. Macromolecular crowding creates heterogeneous environments of gene expression in picolitre droplets
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.
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264. Fibrin-fiber architecture influences cell spreading and differentiation
The mechanical and structural properties of the extracellular matrix (ECM) play an important role in regulating cell fate. The natural ECM has a complex fibrillar structure and shows nonlinear mechanical properties, which are both difficult to mimic synthetically. Therefore, systematically testing the influence of ECM properties on cellular behavior is very challenging. In this work we show two different approaches to tune the fibrillar structure and mechanical properties of fibrin hydrogels. Addition of extra thrombin before gelation increases the protein density within the fibrin fibers without significantly altering the mechanical properties of the resulting hydrogel. On the other hand, by forming a composite hydrogel with a synthetic biomimetic polyisocyanide network the protein density within the fibrin fibers decreases, and the mechanics of the composite material can be tuned by the PIC/fibrin mass ratio. The effect of the changes in gel structure and mechanics on cellular behavior are investigated, by studying human mesenchymal stem cell (hMSC) spreading and differentiation on these gels. We find that the trends observed in cell spreading and differentiation cannot be explained by the bulk mechanics of the gels, but correlate to the density of the fibrin fibers the gels are composed of. These findings strongly suggest that the microscopic properties of individual fibers in fibrous networks play an essential role in determining cell behavior.
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263. Study of thiol-ene chemistry on polymer brushes and application to surface patterning and protein adsorption
Polymer brushes are attractive polymeric coatings for biomedical applications such as tissue engineering, biosensor design and the purification of biological samples. In order to confer bioactivity to these coatings, their functionalisation with biomolecules, proteins and peptides, is required. In this respect, thiolene coupling offers interesting features as it is regioselective, efficient and can be controlled by photoirradiation to allow surface patterning. The efficiency of thiol-ene and the related thiol-yne couplings to poly(glycidyl methacrylate) brushes was quantified. The impact of the structure of thiols to be tethered and reaction conditions on the brush loading and the kinetics of reaction were investigated. Thiol-ene reactions were then used to pattern brushes via direct irradiation through a mask or via reactive microcontact printing. The resulting patterns were used to control albumin adsorption and the formation of protein patterns.
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262. Biocompatible fluorinated polyglycerols for droplet microfluidics as an alternative to PEG-based copolymer surfactants
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.
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261. Dynamic self-organization of side-propelling colloidal rods: experiments and simulations
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 H2O2 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.
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260. Associative Interactions in Crowded Solutions of Biopolymers Counteract Depletion Effects
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.
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259. Influence of Molecular Structure on the Properties of Out-of-Equilibrium Oscillating Enzymatic Reaction Networks
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.
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258. Rational design of functional and tunable oscillating enzymatic networks
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.
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257. Macromolecular Crowding in the Cytosol: Underappreciated or Overestimated?
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256. Deformation of double emulsions under conditions of flow cytometry hydrodynamic focusing
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 degrees 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.
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255. Programmable chemical reaction networks: emulating regulatory functions in living cells using a bottom-up approach
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.
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254. The microenvironment of double emulsions in rectangular microchannels
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 (mu PIV). The multiphase flow system employed in the study had a viscosity ratio,., between aqueous and oil phase of the order of unity (lambda= 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.
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253. One drop at a time: toward droplet microfluidics as a versatile tool for single-cell analysis
Miniaturization has been the key driver for many remarkable technological developments in recent decades. Miniaturization has now also extended into biology, thereby setting the stage for high-throughput single-cell analysis. This advancement is important because, despite detailed molecular information on individual cell subtypes, virtually no information is available on the functional capacities of individual cells. Typical in vivo animal models, as well as in vitro laboratory test tube experiments, only yield a global outcome of interactions of often millions of cells rather than providing insight into the functional contribution of individual cells. Reaction volumes of biological experiments have generally been reduced from milliliters to microliters. Tools and methods that study single-cell behavior have become increasingly important, but often do not allow for high-throughput manipulation. Recent advances in (droplet-based) microfluidics enable systematic high-throughput analyses of individual cells in a highly controlled manner. The implementation of microfluidic technologies in single-cell analysis is one of the most promising approaches that not only offers new information and high-throughput screening but also enables the creation of innovative conditions that are impractical or impossible by conventional methods. In this review, we provide a comprehensive overview of recent developments in droplet-based microfluidics for single-cell studies.
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252. Mechanically strong, fluorescent hydrogels from zwitterionic, fully pi-conjugated polymers
Mechanically strong supramolecular hydrogels (up to 98.9% water content) were obtained by the combination of a rigid, fully pi-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.
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251. DNA-functionalized hydrogels for confined membrane-free in vitro transcription/transtationt
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.
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250. Threshold Sensing through a Synthetic Enzymatic Reaction-Diffusion Network
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.
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249. Interface limited charge extraction and recombination in organic photovoltaics
We report that the transit-time of photogenerated holes within an OPV device is significantly controlled by the interface between the photoactive layer and the electrode. We used time-resolved optical absorption spectroscopy to track the evolution and transport of photogenerated holes in situ within efficient PTB7:PC70BM (thieno [3,4-b] thiophene/benzodithiophene:[6,6]-phenyl C-71-butyric acid methyl ester) devices. Our results demonstrate that the time required for 50% hole extraction is reduced from 2.3 mu s for the standard devices to 0.9 mu s either by introducing a thin layer of conjugated polyelectrolyte (CPE) layer at the electron-extracting cathode or simply by exposing the device to a polar solvent such as methanol which is used for spin coating the CPEs. We consider that the CPE modifies the interface between the photoactive layer and the cathode whereas the exposure of the device to associated polar solvent modifies the buried PEDOT:PSS (poly(3,4-ethylenedioxythiophene) polystyrene sulfonate) hole extracting layer.
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248. Complexity of molecular crowding in cell-free enzymatic reaction networks
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247. Adhesion-Regulated Switchable Fluid Slippage on Superhydrophobic Surfaces
Surface adhesion is regulated by sparsely grafting responsive hydrophilic polymer chains on superhydrophobic surfaces but without obviously changing the wettability. We study experimentally how adhesion of superhydrophobic surfaces affects liquid slip. The slip length of water on such surfaces decays quickly as the adhesive force increases. This intrinsic dependence is theoretically explained based on scaling descriptions for specific geometries. A slip length range of 87 mu m can be achieved reversibly by changing the temperature below and above the low critical solution temperature (LCST) of the grafted temperature-sensitive polymer. The results shed light on the intrinsic mechanism of liquid slip on textured surfaces and have important implications in the design of smart microfluidic and biofluidic devices, in which the regulation of fluid flow is highly desirable.
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246. Vesicle budding from polymersomes templated by microfluidically prepared double emulsions
Many approaches to mimic and understand the dynamics of vesicle budding lack precise control over vesicle membrane properties or require external stimuli to induce budding. We use copolymer-loaded double-emulsion droplets to precisely control size, size distribution, composition and morphology of giant polymersomes. By tuning the copolymer concentration in the polymersome membrane, we identify conditions under which vesicles spontaneously bud from the polymersome surface. Our findings have important implications for the design of copolymermembranes and contribute to the understanding of polymersome formation from double emulsions.
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245. Alterations in Red Blood Cell Deformability during Storage: A Microfluidic Approach
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.
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244. Artificial microniches for probing mesenchymal stem cell fate in 3D
Droplet microfluidics is combined with bio-orthogonal thiol-ene click chemistry to fabricate micrometer-sized, monodisperse fibrinogen-containing hyaluronic acid hydrogel microbeads in a mild, radical-free procedure in the presence of human mesenchymal stem cells (hMSCs). The gel beads serve as microniches for the 3D culture of single hMSCs, containing hyaluronic acid and additional fibrinogen for cell surface binding, and they are porous and stable in tissue culture medium for up to 4 weeks with mechanical properties right in the range of soft solid tissues (0.9-9.2 kPa). The encapsulation procedure results in 70% viable hMSCs in the microbeads after 24 hours of culture and a very high degree of viability of the cells after long term culture of 2 weeks. hMSCs embedded in the microniches display an overall rounded morphology, consistent with those previously observed in 3D culture. Upon induction, the multipotency and differentiation potential of the hMSCs are characterized by staining of corresponding biomarkers, demonstrating a clear heterogeneity in the cell population. These hydrogel microbeads represent a versatile microstructured material platform with great potential for studying the differences of material cues and soluble factors in stem cell differentiation in a 3D tissue-like environment at the single cell level.
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243. An electro-coalescence chip for effective emulsion breaking in droplet microfluidics
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.
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242. Fluorescent hydrogels for studying Ca2+-dependent reaction-diffusion processes
Here, we report a convenient experimental platform to study the diffusion of Ca2+ in the presence of a Ca2+-binding protein (Calbindin D28k). This work opens up new possibilities to elucidate the physical chemistry of complex Ca2+-dependent reaction-diffusion networks that are abundant in living cells.
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241. 25th Anniversary Article: Designer Hydrogels for Cell Cultures: A Materials Selection Guide
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.
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240. Biocompatible macro-initiators controlling radical retention in microfluidic on-chip photopolymerization of water-in-oil emulsions
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.
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239. On the flow topology inside droplets moving in rectangular microchannels
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 (mu PIV). 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.
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238. Learning a New Language: Moving Countries and Changing Subjects
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237. Monodisperse Water-in-Oil-in-Water (W/O/W) Double Emulsion Droplets as Uniform Compartments for High-Throughput Analysis via Flow Cytometry
Here we report the application of monodisperse double emulsion droplets, produced in a single step within partially hydrophilic/partially hydrophobic microfluidic devices, as defined containers for quantitative flow cytometric analysis. Samples with varying fluorophore concentrations were generated, and a clear correlation between dye concentration and fluorescence signals was observed.
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236. Controlled Polymer-Brush Growth from Microliter Volumes using Sacrificial-Anode Atom-Transfer Radical Polymerization
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235. Ultrasensitivity by Molecular Titration in Spatially Propagating Enzymatic Reactions
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.
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234. Role of the extracellular matrix in regulating stem cell fate
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.
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233. Enhanced transcription rates in membrane-free protocells formed by coacervation of cell lysate
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.
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232. Ultrarapid Generation of Femtoliter Microfluidic Droplets for Single-Molecule-Counting Immunoassays
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 x 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.
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231. THE EFFECT OF STORAGE ON RED BLOOD CELL DEFORMABILITY: A MICROFLUIDIC APPROACH
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230. The Influence of Side-Chain Position on the Optoelectronic Properties of a Red-Emitting Conjugated Polymer
A study of the organic semiconductor F8TBT is presented, directly comparing a conventional form (F8TBT-out) with a form with varied alkyl side-chain position (F8TBT-in), in terms of optical properties and device performance in light-emitting-diodes (LEDs). Computational simulations of the side-chain position with respect to the TBT unit reveal geometrical differences between F8TBT-out and F8TBT-in. conjugation on the backbone of F8TBT-in is interrupted by a distortion of the benzothiadiazole ring, leading to a blue-shift of the absorption spectrum and increased photoluminescence quantum efficiency. Both conventional and hybrid LEDs demonstrate that devices with F8TBT-in show improved performance, as compared to F8TBT-out, illustrating how tuning the optoelectronic properties of conjugated polymers by varying the placement of side chains has an important role in device optimization.
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229. Cooperative Adsorption of Lipoprotein Phospholipids, Triglycerides, and Cholesteryl Esters Are a Key Factor in Nonspecific Adsorption from Blood Plasma to Antifouling Polymer Surfaces
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.
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228. Donor-acceptor interface modification by zwitterionic conjugated polyelectrolytes in polymer photovoltaics
We report that we can enhance the performance of polymer photovoltaics by introducing materials with dipoles at the donor-acceptor interface of the polymer solar cells. We use a zwitterionic conjugated polyelectrolyte (ZCPE) as an interfacial layer in a model bi-layer system consisting of donor and acceptor polymers. We find an increase in short-circuit current and fill factor when a 2 nm thin layer of ZCPE is introduced between donor and acceptor polymers. Absorption of light in the ZCPE interlayer contributes to the photocurrent, and, as revealed by time-resolved pump-push optical spectroscopy, the interlayer reduces the probability of electron-hole recombination at the donor-acceptor interface.
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227. Intra-Species Bacterial Quorum Sensing Studied at Single Cell Level in a Double Droplet Trapping System
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.
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226. Sensitive, High Throughput Detection of Proteins in Individual, Surfactant-Stabilized Picoliter Droplets Using Nanoelectrospray Ionization Mass Spectrometry
Droplet-based fluidics is emerging as a powerful platform for single cell analysis, directed evolution of enzymes, and high throughput screening studies. Due to the small amounts of compound compartmentalized in each droplet, detection has been primarily by fluorescence. To extend the range of experiments that can be carried out in droplets, we have developed the use of electrospray ionization mass spectrometry (ESI-MS) to measure femtomole quantities of proteins in individual pico- to nanoliter droplets. Surfactant-stabilized droplets containing analyte were produced in a flow-focusing droplet generation microfluidic device using fluorocarbon oil as the continuous phase. The droplets were collected off-chip for storage and reinjected into microfluidic devices prior to spraying the emulsion into an ESI mass spectrometer. Crucially, high quality mass spectra of individual droplets were obtained from emulsions containing a mixture of droplets at >150 per minute, opening up new routes to high throughput screening studies.
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225. Control of Intrachain Charge Transfer in Model Systems for Block Copolymer Photovoltaic Materials
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.
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224. Liquid crystalline chromophores for photonic band-edge laser devices
We present results on laser action from liquid crystal compounds whereby one sub-unit of the molecular structure consists of the cyano-substituted chromophore, {phenylene-bis (2-cyanopropene)}, similar to the basic unit of the semiconducting polymer structure poly(cyanoterephthalylidene). These compounds were found to exhibit nematic liquid crystal phases. In addition, by virtue of the liquid crystalline properties, the compounds were found to be highly miscible in wide temperature range commercial nematogen mixtures. When optically excited at lambda = 355 nm, laser emission was observed in the blue/green region of the visible spectrum (480-530 nm) and at larger concentrations by weight than is achievable using conventional laser dyes. Upon increasing the concentration of dye from 2 to 5 wt.% the threshold was found to increase from E-th = 0.42 +/- 0.02 mu J/pulse (approximate to 20 mJ/cm(2)) to E-th = 0.66 +/- 0.03 mu J/pulse (approximate to 34 mJ/cm(2)). Laser emission was also observed at concentrations of 10 wt.% but was less stable than that observed for lower concentrations of the chromophore. (c) 2012 Elsevier B.V. All rights reserved.
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223. Panchromatic Dye-Doped Polymer Solar Cells: From Femtosecond Energy Relays to Enhanced Photo-Response
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)-4Hcyclopenta[2,1-b;3,4-b0]clithiophene)-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-yObenzo[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 photoresponse in a broad range of polymeric and hybrid solar cells.
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222. Electrochemically Mediated Atom Transfer Radical Polymerization on Nonconducting Substrates: Controlled Brush Growth through Catalyst Diffusion
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 [(CuL)-L-II]/[(CuL)-L-I] 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 [(CuL)-L-II]/[(CuL)-L-I] gradient.
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221. Monodisperse collagen-gelatin beads as potential platforms for 3D cell culturing
A droplet-based microfluidics technique is used to produce monodisperse, 80 mm collagen-gelatin beads with tunable mechanical properties in the range of 1-10 kPa after photo-crosslinking. The gel beads are porous, mechanically robust and stable in buffer, but can be degraded enzymatically. Encapsulated fibroblast cells maintain 70% viability after one-week encapsulation and preliminary results show that the degree of spreading of cells in gels is correlated with the stiffness of the material.
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220. Decoupling geometrical and chemical cues directing epidermal stem cell fate on polymer brush-based cell micro-patterns
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 zeta-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).
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219. All-polymer field-effect transistors using a brush gate dielectric
Interfaces between a poly(3-hexylthiophene) [P3HT] and an end-grafted (brush) layer of poly(methyl methacrylate) [PMMA] are shown using neutron reflectometry to be dependent on heat treatment. Annealing the samples allows part of the brush layer to cross into the P3HT layer creating a very asymmetric interface. We suggest that the P3HT rearrangement occurs, creating space for movement of the brush into the film. This interpenetration was observed with two different molecular weight (17.5 and 28 kg mol(-1)) P3HT films. Output characteristics of devices made from P3HT layers on PMMA brushes show that different amounts of heat treatment do not significantly change the device performance. Saturated hole mobilities are dependent on heat treatment, with devices made from a smaller molecular weight P3HT (22 kg mol(-1)) demonstrating larger mobilities than devices created using 48 kg mol(-1) P3HT, but only after heat treatment.
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218. Probing cellular heterogeneity in cytokine-secreting immune cells using droplet-based microfluidics
Here, we present a platform to detect cytokine (IL-2, IFN-gamma, TNF-alpha) 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.
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217. Self-organization of the bacterial cell-division protein FtsZ in confined environments
We report a microfluidic approach to generate aqueous droplets in oil of different dimensionality, stabilized by a lipid monolayer, to systematically probe the polymerization of bacterial cell-division protein FtsZ into fibrous networks as a function of the concentrations of crowding agent, FtsZ, and GTP. FtsZ bundles confined in droplets were dynamic, and their distribution depended on the intrinsic properties of the system and restrictions imposed by the spatial boundaries.
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216. Nonfouling Capture-Release Substrates Based on Polymer Brushes for Separation of Water-Dispersed Oil Droplets
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.
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215. Synthesis and Photophysics of Fully pi-Conjugated Heterobis-Functionalized Polymeric Molecular Wires via Suzuki Chain-Growth Polymerization
We present a fast and efficient in situ synthetic approach to obtain fully pi-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 H-1 and NMR. End group fiinctionalization 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.
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214. Fabrication of Microgel Particles with Complex Shape via Selective Polymerization of Aqueous Two-Phase Systems
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213. Extracellular-matrix tethering regulates stem-cell fate (vol 11, pg 642, 2012)
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212. Mimicking normal tissue architecture and perturbation in cancer with engineered micro-epidermis
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 mu m 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 mu m 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. (C) 2012 Elsevier Ltd. All rights reserved.
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211. Extracellular-matrix tethering regulates stem-cell fate
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.
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210. Formation of Spherical and Non-Spherical Eutectic Gallium-Indium Liquid-Metal Microdroplets in Microfluidic Channels at Room Temperature
Here, the formation of eutectic Gallium-Indium (EGaIn) liquid-metal microdroplets, both spherical and non-spherical, in microfluidic devices at room temperature is reported. Monodisperse microdroplets were created in an aqueous polyethylene glycol (PEG) solution, in oxygenated and in deoxygenated silicone oil. The volume of the droplets depends on the channel dimensions and flow rates applied, varying between 0.5 and 4 nL. Non-spherical droplets were formed in oxygenated silicone oil due to the instantaneous formation of an oxide layer. These metal micro-rice droplets retained their shape and did not spontaneously reflow to form shapes of the lowest interfacial energy on egress from the channel, unlike in aqueous PEG solution and in deoxygenated silicone oil. Liquid-metal droplets with such tunable morphology can potentially be used in MEMS devices for optical and electrical switches, valves and micropumps.
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209. Synthesis, Purification, and Characterization of Well-Defined All-Conjugated Diblock Copolymers PF8TBT-b-P3HT
We present the synthesis, purification, and characterization of all-conjugated block copolymers comprising poly((9,9-dioctylfluorene)-2,7-diyl-alt-[4,7-bis(3-hexylthien-5-yl)- 2,1,3-benzothiadiazole]-2′,2 ”-diyl) (PF8TBT) and poly(3-hexylthiophene) (P3HT). Suzuki step-growth polycondensation is used for the synthesis of PF8TBT, which is subsequently terminated via the addition of narrow-distributed, monobrominated P3HT-Br. Purification via preparative GPC is carried out to reduce polydispersity and to remove excess P3HT. Wavelength-dependent GPC and careful NMR end group analysis, assisted by model compounds, reveal pure diblock copolymers of PF8TBT-b-P3HT. Insight into structure formation is given by temperature-dependent UV-vis absorption, DSC, and X-ray scattering. These indicate that PF8TBT-b-P3HT does not microphase-separate within the investigated range of composition and molecular weight. The critical role of introducing sufficient dissimilarity between the segments in all-conjugated block copolymers in order to induce phase separation is discussed, with the conclusion that careful tuning of side chains is crucial for achieving self-organization.
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208. On the Role of Single Regiodefects and Polydispersity in Regioregular Poly(3-hexylthiophene): Defect Distribution, Synthesis of Defect-Free Chains, and a Simple Model for the Determination of Crystallinity
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)Cl-2 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,similar to 70%. For Ni(dppp)Cl-2-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.
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207. Structure and Collapse of a Surface-Grown Strong Polyelectrolyte Brush on Sapphire
We have used neutron reflectometry to investigate the behavior of a strong polyelectrolyte brush on a sapphire substrate, grown by atom-transfer radical polymerization (ATRP) from a silane-anchored initiator layer. The initiator layer was deposited from vapor, following treatment of the substrate with an Ar/H2O plasma to improve surface reactivity. The deposition process was characterized using X-ray reflectometry, indicating the formation of a complete, cross-linked layer. The brush was grown from the monomer [2-(methacryloyloxy)ethyl]trimethyl-ammonium chloride (METAC), which carries a strong positive charge. The neutron reflectivity profile of the swollen brush in pure water (D2O) showed that it adopted a two-region structure, consisting of a dense surface region similar to 100 angstrom thick, in combination with a diffuse brush region extending to around 1000 angstrom from the surface. The existence of the diffuse brush region may be attributed to electrostatic repulsion from the positively charged surface region, while the surface region itself most probably forms due to polyelectrolyte adsorption to the hydrophobic initiator layer. The importance of electrostatic interactions in maintaining the brush region is confirmed by measurements at high (1 M). added 1:1 electrolyte, which show a substantial transfer of polymer from the brush to the surface region, together with a strong reduction in brush height. On addition of 10(-4) M oppositely charged surfactant (sodium dodecyl sulfate), the brush undergoes a dramatic collapse, forming a single dense layer about 200 angstrom in thickness, which may be attributed to the neutralization of the monomers by adsorbed dodecyl sulfate ions in combination with hydrophobic interactions between these dodecyl chains. Subsequent increases in surfactant concentration result in slow increases in brush height, which may be caused by stiffening of the polyelectrolyte chains due to further dodecyl sulfate adsorption.
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206. Oligomeric Compatibilizers for Control of phase Separation in Conjugated Polymer Blend Films
Control over phase separation and morphology is critical to optimal function in polymer optoelectronic devices. Here, two fully conjugated oligomeric compatibilizers are introduced, and their effect on the phase separation of blends of poly(9,9′-dioctylfluorene-co-benzo-thiadiazole) (F8BT) with poly(9,9′-dioctylfluorene-co-bis-N,N’-(4,butylphenyl)bis-N,N’-phenyl-1,4-phenylenediamine) (PFB) are reported. AFM and STXM analysis demonstrate that the addition of compatibilizer altered the size and relative composition of phase-separated domains formed during spin-casting. Small structural differences between the two compatibilizers brought about significantly different morphological changes to the blends, suggesting that further development of compatibilizer structure could enable enhanced control toward desired blend film morphologies.
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205. Single Molecule Fluorescence under Conditions of Fast Flow
We have experimentally determined the optimal flow velocities to characterize or count single molecules by using a simple microfluidic device to perform two-color coincidence detection (TCCD) and single pair Forster resonance energy transfer (spFRET) using confocal fluorescence spectroscopy on molecules traveling at speeds of up to 10 cm s(-1). We show that flowing single fluorophores at >= 0.5 cm s(-1) reduces the photo-physical processes competing with fluorescence, enabling the use of high excitation irradiances to partially compensate for the short residence time within the confocal volume (10-200 mu s). Under these conditions, the data acquisition rate can be increased by a maximum of 38-fold using TCCD at 5 cm s(-1) or 18-fold using spFRET at 2, cm s(-1), when compared with diffusion. While structural characterization requires more photons to be collected per event and so necessitates the use of slower speeds (2 cm s(-1) for TCCD and 1 cm s(-1) for spFRET), a considerable enhancement in the event rate could still be obtained (33-fold for TCCD and 16-fold for spFRET). Using flow under optimized conditions, analytes could be rapidly quantified over a dynamic range of up to 4 orders of magnitude by direct molecule counting; a 50 fM dual-labeled model sample can be detected with 99.5% statistical confidence in around 8 s using TCCD and a flow velocity of 5 cm s(-1).
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204. Investigation of On Water Conditions Using a Biphasic Fluidic Platform
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203. Electrochemically Induced Surface-Initiated Atom-Transfer Radical Polymerization
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202. Microfluidic platform for combinatorial synthesis in picolitre droplets
This paper presents a droplet-based microfluidic platform for miniaturized combinatorial synthesis. As a proof of concept, a library of small molecules for early stage drug screening was produced. We present an efficient strategy for producing a 7 x 3 library of potential thrombin inhibitors that can be utilized for other combinatorial synthesis applications. Picolitre droplets containing the first type of reagent (reagents A(1), A(2), …, A(m)) were formed individually in identical microfluidic chips and then stored off chip with the aid of stabilizing surfactants. These droplets were then mixed to form a library of droplets containing reagents A(1-m), each individually compartmentalized, which was reinjected into a second microfluidic chip and combinatorially fused with picolitre droplets containing the second reagent (reagents B-1, B-2, …, B-n) that were formed on chip. The concept was demonstrated with a three-component Ugi-type reaction involving an amine (reagents A(1-3)), an aldehyde (reagents B1-7), and an isocyanide (held constant), to synthesize a library of small molecules with potential thrombin inhibitory activity. Our technique produced 10(6) droplets of each reaction at a rate of 2.3 kHz. Each droplet had a reaction volume of 3.1 pL, at least six orders of magnitude lower than conventional techniques. The droplets can then be divided into aliquots for different downstream screening applications. In addition to medicinal chemistry applications, this combinatorial droplet-based approach holds great potential for other applications that involve sampling large areas of chemical parameter space with minimal reagent consumption; such an approach could be beneficial when optimizing reaction conditions or performing combinatorial reactions aimed at producing novel materials.
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201. Influence of Side Chains on Geminate and Bimolecular Recombination in Organic Solar Cells
The effects of the side chains on the loss processes in three polymer:PC[61]BM organic blends are studied by comparing transient absorption spectroscopy and solar cell measurements. It is shown that, after efficient photoinduced charge transfer, charge pairs are the only significant species present in the blend and that their recombination can be followed by monitoring the PIA kinetics. Charges are found to decay following two regimes. The subnanosecond decay of the charge signal, responsible for the losses of 55-75% of the charges, is well described by an Onsager-Braun geminate recombination model. The rates are found to be strongly affected by the presence and position of side chains, We attribute these changes to a better aggregation of the side-chain-less polymer that provides a better delocalization of the holes and a better mobility. At longer times (after about 10 ns) the charges undergo bimolecular recombination. By modeling the time and intensity dependence of this process, we measure the rate constant and find that it is similar for the three systems and slightly lower than the theoretical Langevin rate. By comparing these spectroscopic results with the light-intensity and bias dependence of the photocurrent, we conclude that bimolecular recombination does not play an important role under solar intensity. Geminate recombination is the main loss mechanism and explains the difference between all the devices, their strong bias dependence, and their generally low performance.
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200. Chain-Growth Polymerization of Unusual Anion-Radical Monomers Based on Naphthalene Diimide: A New Route to Well-Defined n-Type Conjugated Copolymers
Strongly electron-deficient (n-type) main-chain g-conjugated polymers are commonly prepared via well-established step-growth polycondensation protocols which enable limited control over polymerization. Here we demonstrate that activated Zn and electron-deficient brominated thiophene-naphthalene diimide oligomers form anion-radical complexes instead of conventional Zn-organic derivatives. These highly unusual zinc complexes undergo Ni-catalyzed chain-growth polymerization leading to n-type conjugated polymers with controlled molecular weight, relatively narrow polydispersities, and specific end-functions.
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199. Ring Walking versus Trapping of Nickel(0) during Kumada Catalyst Transfer Polycondensation Using Externally Initiated Electron-Accepting Thiophene-Benzothiadiazole-Thiophene Precursors
Interactions of Ni(0) and thiophene benzothiadiazole-thiophene (TBT) units during the Kumada catalyst transfer polycondensation (KCTP) of 2-chloromagnesio-5-bromo-3-hexylthiophene (1) are investigated. Monofunctional TBT initiator precursors are used for the external initiation of KCTP, and the mechanism of initiator activation and polymerization is elucidated using NMR and MALDI-ToF MS. We find that the activation of the TBT-bromide initiator precursor using nickel-diethylbipyridine (NiEt(2)bipy) occurs via a two-step pathway, in which NiEt(2)bipy coordinates to benzothiadiazole (B) first, followed by the elimination of butane and oxidative addition of liberated Ni(0)bipy into the terminal TBT-Br bond. It is shown that the B unit traps Ni(0) during the KCTP of 1, which results in significant termination, as derived from the degree of bromine-terminated chains. The ability of B units to trap Ni(0) is further illustrated by using a symmetric bifunctional Br-TBT-Br initiator precursor for the KCTP of 1, during which Ni(0) is not able to ring walk over the B unit to initiate polymerization at the other end of the chain. These results are important for the design of well-defined and electronically end-functionalized conjugated polymers, but also for understanding termination mechanisms in KCTP in general.
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198. Chain-Growth Suzuki Polymerization of n-Type Fluorene Copolymers
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197. Effect of Polymer Brush Architecture on Antibiofouling Properties
Polymer brushes show great promise in next-generation antibiofouling surfaces. Here, we have studied the influence of polymer brush architecture on protein resistance. By carefully optimizing reaction conditions, we were able to polymerize oligoglycerol-based brushes with sterically demanding linear or dendronized side chains on gold surfaces. Protein adsorption from serum and plasma was analyzed by surface plasmon resonance. Our findings reveal a pronounced dependence of biofouling on brush architecture. Bulky yet flexible side chains as in dendronized brushes provide an ideal environment to repel protein-possibly through formation of a hydration layer, which can be further enhanced by presenting free hydroxyl groups on the polymer brushes. A deeper understanding of how brush architecture influences protein resistance will ultimately enable fabrication of surface coatings tailored to specific requirements in biomedical applications.
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196. Polymer Brushes Showing Non-Fouling in Blood Plasma Challenge the Currently Accepted Design of Protein Resistant Surfaces
Ultra-low-fouling poly[N-(2-hydroxypropyl) methacrylamide] (poly(HPMA)) brushes have been synthesized for the first time. Similar to the so far only ultra-low-fouling surface, poly(carboxybetaine acrylamide), the level of blood plasma fouling was below the detection limit of surface plasmon resonance (SPR, 0.03 ng . cm(-2)) despite being a hydrogen bond donor and displaying a moderate wettability, thus challenging the currently accepted views for the design of antifouling properties. The antifouling properties were preserved even after two years of storage. To demonstrate the potential of poly( HPMA) brushes for the preparation of bioactive ultra-low fouling surfaces a label-free SPR immunosensor for detection of G Streptococcus was prepared.
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195. MATERIALS CHEMISTRY Polymer networks take a bow
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194. Tuning the electronic coupling in a low-bandgap donor-acceptor copolymer via the placement of side-chains
We present a spectroscopic and theoretical investigation of the effect of the presence and position of hexyl side-chains in the novel low-bandgap alternating donor-acceptor copolymer poly[bis-N,N-(4-octylphenyl)-bis-N,N-phenyl-1,4-phenylenediamine-alt-5,5′-4′,7′,-di-2-thienyl-2′,1′,3′-benzothiadiazole] (T8TBT). We use electronic absorption and Raman spectroscopic measurements supported by calculations of chain conformation, electronic transitions, and Raman modes. Using these tools, we find that sterically demanding side-chain configurations induce twisting in the electronic acceptor unit and reduce the electronic interaction with the donor. This leads to a blue-shifted and weakened (partial) charge-transfer absorption band together with a higher photoluminescence efficiency. On the other hand, sterically relaxed side-chain configurations promote coupling between donor and acceptor units and exhibit enhanced absorption at the expense of luminescence efficiency. The possibility of tuning the donor-acceptor character of conjugated polymers by varying the placement of side-chains has very important ramifications for light emitting diode, Laser, display, and photovoltaic device optimization. (C) 2011 American Institute of Physics. [doi:10.1063/1.3562936]
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193. Enhanced charge transport by incorporating additional thiophene units in the poly(fluorene-thienyl-benzothiadiazole) polymer
We report a comparative study of optical properties, structure and morphology, field-effect transistor (FET) and solar cell performance between poly(4-(3,4′-dihexyl-2,2′-bithiophen-5-yl)-7-(5′-(9,9-dioctyl-9H-fluoren-2-yl)-3,4′-dihexyl-2,2′-bithiophen-5-yl)benzo[c][1,2,5]-thiadiazole) (F8TTBTT), and its predecessor poly((9,9-dioctylfluorene)-2,7-diyl-alt[4,7-bis(3-hexylthien-5-yl)-2,1,3-benzothiadiazole]-2′,2 ”-diyl) (F8TBT). Compared to F8TBT, F8TTBTT has two more thiophene units incorporated in its monomer structure. Such a modification leads to a reduced optical band gap, improved charge injection and significantly enhanced ambipolar field-effect mobilities reaching 5 x 10 (2) cm(2) V (1) s (1) for holes and 4 x 10 (3) cm(2) V (1) s (1) for electrons. The enhanced carrier mobilities are most likely a result of an increased backbone planarization and interchain interaction. As a consequence of ambipolar transport, light-emission was observed from the transistor channel during operation. The reduced band gap and improved charge transport make F8TTBTT an interesting candidate also for solar cell applications. Unoptimized solar cells based on F8TTBTT: PCBM blends were found to exhibit power conversion efficiency under AM 1.5 illumination of similar to 1.54%. (C) 2011 Published by Elsevier B.V.
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192. Controlled Folding of 2D Au-Polymer Brush Composites into 3D Microstructures
Microscale, quasi-2D Au-polymer brush composite objects are fabricated by a versatile, controllable process based on microcontact printing followed by brush growth and etching of the substrate. These objects fold into 3D microstructures in response to a stimulus: crosslinked poly(glycidyl methacrylate) (PGMA) brushes fold on immersion in MeOH, and poly(methacryloxyethyl trimethylammonium chloride) (PMETAC) brushes fold on addition of salt. Microcages and microcontainers are fabricated. A multistep microcontact printing process is also used to create sheets of Au-PGMA bilayer lines linked by a PGMA film, which fold into cylindrical tubes. The bending of these objects can be predicted, and hence predefined during the synthesis process by controlling the parameters of the gold layer, and of the polymer brush.
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191. Formation of Pickering Emulsions Using Ion-Specific Responsive Colloids
The ability to control the dispersion, aggregation, and assembly of colloidal systems is important for a number of applications, for instance, Pickering emulsions, drug and gene delivery, control of fluid rheology, and the formation of colloidal crystal arrays. We generated a responsive colloidal system based on polymer-brush-grafted silica nanoparticles and demonstrated that such a colloidal system can be used to produce stable oil-in-water Pickering emulsions. Cationic poly(2-(methacryloyloxy)-ethyl-trimethyl-ammonium chloride) (PMETAC) brushes were grown from silica nanopartides (diameter similar to 320 nm) through surface-initiated atom-transfer radical polymerization (ATRP). PMETAC brushes are attractive coatings for controlling the behavior of colloidal systems, owing to their ion-specific collapse resulting in the switching of surface hydrophilicity. Fourier transform infrared (FT-IR) spectroscopy, thermogravimetric analysis (TGA), transmission electron microscopy (TEM), dynamic light scattering (DLS), and zeta-potential measurements indicated the successful grafting of PMETAC brushes on nanoparticles. The resulting colloidal dispersion was shown to be responsive to perchlorate ions (ClO4-), which triggered particle aggregation and enabled the generation of Pickering emulsions. The onset of aggregation depended on the polymer chain length. Aggregation was not affected by the initiator density and brush conformational changes. Further studies suggested that particle aggregation and the formation of stable Pickering emulsions were not simply due to brush collapse but also were due to a gradual shielding of electrostatic repulsion. Finally, the stability and homogeneity of the resulting Pickering emulsions were studied
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190. Monitoring a Reaction at Submillisecond Resolution in Picoliter Volumes
Well-established rapid mixing techniques such as stopped-flow have been used to push the dead time for kinetic experiments down to a few milliseconds. However, very fast reactions are difficult to resolve below this limit. We now outline an approach that provides access to ultrafast kinetics but does not rely on active mixing at all. Here, the reagents are compartmentalized into water-in-oil emulsion microdroplets (diameter similar to 50 mu m) that are statically arrayed in pairs, resting side-by-side in a well feature of a poly(dimethylsiloxane) (PDMS) device. A reaction between the contents of two droplets arrayed in such a holding trap is initiated by droplet fusion that is brought about by electrocoalescence and known to occur on a time scale of about 100 mu s. A reaction between the reactants (Fe3+ and SCN-) is monitored by image analysis measuring the product formation in the newly merged drop in both space and time, by use of a fast camera. A comparison of the concentration field of the reaction product with the output of a reaction-diffusion system of equations yields a rate constant k similar to 3 x 10(4) M-3.s(-1). Since reaction and diffusion are formally included in the mathematical model, measurements can proceed immediately after the drop fusion, removing the need to allow time for mixing. This approach is different from existing methodologies, for example, experiments in a conventional stopped-flow apparatus but also electrofusion of moving droplets where contents are mixed by chaotic advection before a reaction is monitored. Our analysis makes kinetics accessible that are several times faster than techniques that have to allow time for mixing.
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189. Conjugated Zwitterionic Polyelectrolyte as the Charge Injection Layer for High-Performance Polymer Light-Emitting Diodes
A new zwitterionic conjugated polyelectrolyte without free counterions has been used as an electron injection material in polymer light-emitting diodes. Both the efficiency and maximum brightness were considerably improved in comparison with standard Ca cathode devices. The devices showed very fast response times, indicating that the improved performance is, in addition to hole blocking, due to dipoles at the cathode interface, which facilitate electron injection.
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188. Formation of Well-Ordered Heterojunctions in Polymer: PCBM Photovoltaic Devices
The nanoscale morphology in polymer:PCBM based photovoltaic devices is a major contributor to overall device performance. The disordered nature of the phase-separated structure, in combination with the small length scales involved and the inherent difficulty of reproducing the exact morphologies when spin-coating and annealing thin blend films, have greatly hampered the development of a detailed understanding of how morphology impacts photo voltaic device functioning. In this paper we demonstrate a double nanoimprinting process that allows the formation of nanostructured polymer: PCBM heterojunctions of composition and morphology that can be selected independently. We fabricated photovoltaic (PV) devices with extremely high densities (10(14) mm(-2)) of interpenetrating nanoscale columnar features (as small as 25 nm; at or below the exciton diffusion length) in the active layer. By comparing device results of different feature sizes and two different polymer: PCBM combinations, we demonstrate how double imprinting can be a powerful tool to systematically study different parameters in polymer photovoltaic devices.
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187. Island brushes to control adhesion of water in oil droplets on planar surfaces
By using molecular self-assembly and polymer brush chemistry, adhesion of water droplets at solid/oil interfaces could be achieved and modulated by external triggers. Silicon wafers were hydrophobically modified with binary mixed self-assembled layers consisting of fluorinated silanes and atom transfer radical polymerisation (ATRP) initiator silanes, and subsequently grafted with responsive polymers via surface-initiated ATRP. Temperature-and pH-responsive adhesion of water in oil droplets occurred on surfaces coated with phase-separated self-assembled layers and grafted with short polymer chains.
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186. Direct Correlation between Local Pressure and Fluorescence Output in Mechanoresponsive Polyelectrolyte Brushes
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185. Quantitative tracking of the growth of individual algal cells in microdroplet compartments
In this paper we introduce a simple droplet-based microfluidic system consisting of two separate devices to encapsulate and culture microalgae, in contrast to cultivation in bulk liquid medium. This microdroplet technology has been used to monitor the growth of individual microalgal cells in a constant environment for extended periods of time. Single cells from three species of green microalgae, (two freshwater species Chlamydomonas reinhardtii and Chlorella vulgaris, and one saline species Dunaliella tertiolecta), were encapsulated and incubated in microdroplet compartments of diameter of similar to 80 mu m, and their growth analysed over 10 days. In all cases, the doubling time of microalgae grown in microdroplets was similar to growth in bulk. The growth of C. reinhardtii in microdroplets of varying diameters and with different initial cell numbers per droplet was investigated, as well as the effect of varying medium conditions such as pH and nitrogen concentration. This methodology offers the opportunity to study characteristics over time of individual cells and colonies, as well as to screen large numbers of them.
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184. Controlling the contents of microdroplets by exploiting the permeability of PDMS
A microfluidic device capable of exploiting the permeability of small molecules through polydimethylsiloxane (PDMS) has been fabricated in order to control the contents of microdroplets stored in storage wells. We demonstrate that protein precipitation and crystallization can be triggered by delivery of ethanol from a reservoir channel, thus controlling the protein solubility in microdroplets. Likewise quorum sensing in bacteria was triggered by delivery of the auto-inducer N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL) through the PDMS membrane of the device.
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183. Microdroplet fabrication of silver-agarose nanocomposite beads for SERS optical accumulation
Microdroplets have been used as reactors for the fabrication of agarose beads with high uniformity in shape and size, and densely loaded with silver ions, which were subsequently reduced into nanoparticles using hydrazine. The resulting nanocomposite beads not only display a high plasmonic activity, but can also trap/concentrate analytes, which can be identified by means of surface-enhanced Raman scattering (SERS) spectroscopy. The size of the beads is such that it allows the detection of a single bead under a conventional optical microscope, which is very useful to reduce the amount of material required for SERS detection.
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182. Microfluidic production of monodisperse functional o/w droplets and study of their reversible pH dependent aggregation behavior
We report the use of microfluidics for the production of monodisperse oil-in-water droplets functionalized by a pH responsive branched co-polymer surfactant. The droplet functionality facilitates the reversible aggregation of the micron-sized droplets into macroscopic engineered emulsions in response to solution pH changes. Co-injection of dye-loaded and non-dyed droplets into acidic water yields bi-colored dumbbell-shaped aggregates that disassemble into their constituent droplet building blocks upon an increase in pH. Optical tweezers are used to study and quantify the pH dependent interactions of individual droplets.
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181. Convenient Route To Initiate Kumada Catalyst-Transfer Polycondensation Using Ni(dppe)Cl-2 or Ni(dppp)Cl-2 and Sterically Hindered Grignard Compounds
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180. Effect of self-complementary motifs on phase compatibility and material properties in blends of supramolecular polymers
Applicability of supramolecular polymers blending in preparation of materials with tunable properties is reported. The effect of strongly dimerising 2-ureido-4[1H]-pyrimidinone (UPy) end groups on phase compatibility in binary polymer blends was studied. A low molecular weight poly(tetrahydrofuran) diol was functionalized with UPy moieties (PTHF(UPy)(2), a soft material) and mixed with varying amounts of low molecular weight UPy functionalized polycaprolactone di- and triol (PCL(UPy)(2) and PCL(UPy)(3), both relatively strong and stiff materials). Thermal studies showed that T-g of the homopolymers shifted to intermediate temperatures. AFM observation suggested that the phase domain decreased significantly after UPy functionalization of diols. Also the mechanical properties improved at a higher rate than those predicted by the rule of mixtures. These findings indicate that blend components are placed in intimate contact as a result of the UPy UPy interactions. The reversible crosslinking by PCL(UPy)(3) gave better control still over the mechanical properties of the supramolecular polymer blends. (C) 2010 Elsevier Ltd. All rights reserved.
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179. Enhanced Photoresponse in Solid-State Excitonic Solar Cells via Resonant Energy Transfer and Cascaded Charge Transfer from a Secondary Absorber
We present a spiro linked molecule 2 2′ 7 7′ tetrakis(3 hexyl 5 (7 (4 hexylthlophen 2 yl)benzo[c][1 2 5]thiadiazol 4 4 yl)thiophen 2 yl) 9 9′ spirobifluorene which acts as a secondary absorber in solid state excitonic solar cells Blending with a hole transpoting matenal 2 2’7 7′-tetrakis(N N di-p-methoxyphenyl amine) 9 9′ spirobifluorene and used in conjunction with a near infrared dye (termed TTI) results in an extended spectral response which yields a notable increase in short circuit current and power conversion efficiency This enhancement is due to both exciton energy transfer and also nanoscale charge generation in the blend via the formation of an excited sate spiro complex with charge transfer character
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178. Biological and mechanical properties of novel composites based on supramolecular polycaprolactone and functionalized hydroxyapatite
Supramolecular polymers based on quadruple hydrogen-bonding ureido-pyrimidinone (UPy) moieties hold promise as dynamic/stimuli-responsive materials in applications such as tissue engineering. Here, a new class of materials is introduced: supramolecular polymer composites. We show that despite the highly ordered structure and tacticity-dependent nature of hydrogen-bonded supramolecular polymers, the bioactivity of these polymers can be tuned through composite preparation with bioceramics. These novel supramolecular composites combine the superior processability of supramolecular polymers with the excellent bioactivity and mechanical characteristics of bioceramics. In particular, the bioactive composites prepared from supramolecular polycaprolactone and UPy-grafted hydroxyapatite (HApUPy) are described that can be easily formed into microporous biomaterials. The compression moduli increased about 40 and 90% upon composite preparation with HAp and HApUPy, respectively, as an indication to improved mechanical properties. These new materials show excellent potential as microporous composite scaffolds for the adhesion and proliferation of rat mesenchymal stem cells (rMSCs) as a first step toward bone regeneration studies; rMSCs proliferate about 2 and 2.7 times faster on the conventional composite with HAp and the supramolecular composite with (HApUPy) than on the neat PCL1250(UPy)2. (C) 2010 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 95A: 209-221, 2010.
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177. Fabrication of Sub-10 nm Metallic Lines of Low Line-Width Roughness by Hydrogen Reduction of Patterned Metal-Organic Materials
The fabrication of very narrow metal lines by the lift-off technique, especially below sub-10 nm, is challenging due to thinner resist requirements in order to achieve the lithographic resolution. At such small length scales, when the grain size becomes comparable with the line-width, the built-in stress in the metal film can cause a break to occur at a grain boundary. Moreover, the line-width roughness (LWR) from the patterned resist can result in deposited metal lines with a very high LWR, leading to an adverse change in device characteristics. Here a new approach that is not based on the lift-off technique but rather on low temperature hydrogen reduction of electron-beam patterned metal naphthenates is demonstrated. This not only enables the fabrication of sub-10 nm metal lines of good integrity, but also of low LWR, below the limit of 3.2 nm discussed in the International Technology Roadmap for Semiconductors. Using this method, sub-10 nm nickel wires are obtained by reducing patterned nickel naphthenate lines in a hydrogen-rich atmosphere at 500 degrees C for 1 h. The LWR (Le., 3 sigma(LWR)) of these nickel nanolines was found to be 2.9 nm. The technique is general and is likely to be suitable for fabrication of nanostructures of most commonly used metals (and their alloys), such as iron, cobalt, nickel, copper, tungsten, molybdenum, and so on, from their respective metal-organic compounds.
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176. Actin and serum response factor transduce physical cues from the microenvironment to regulate epidermal stem cell fate decisions
Epidermal homeostasis depends on a balance between stem cell renewal and differentiation and is regulated by extrinsic signals from the extracellular matrix (ECM)(1,2). A powerful approach to analysing the pathways involved is to engineer single-cell microenvironments in which individual variables are precisely and quantitatively controlled(3-5). Here, we employ micropatterned surfaces to identify the signalling pathways by which restricted ECM contact triggers human epidermal stem cells to initiate terminal differentiation. On small (20 mu m diameter) circular islands, keratinocytes remained rounded, and differentiated at higher frequency than cells that could spread on large (50 mu m diameter) islands. Differentiation did not depend on ECM composition or density. Rather, the actin cytoskeleton mediated shape-induced differentiation by regulating serum response factor (SRF) transcriptional activity. Knockdown of SRF or its co-factor MAL inhibited differentiation, whereas overexpression of MAL stimulated SRF activity and involucrin expression. SRF target genes FOS and JUNB were also required for differentiation: c-Fos mediated serum responsiveness, whereas JunB was regulated by actin and MAL. Our findings demonstrate how biophysical cues are transduced into transcriptional responses that determine epidermal cell fate.
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175. Controlled Bending of Microscale Au-Polyelectrolyte Brush Bilayers
Free-standing Au-polyelectrolyte brush bilayer objects were fabricated by a facile route based on microcon tact printing and chemical etching. Patterned poly(methacryloxyethyltrimethylammonium chloride) (PMETAC) brushes were grown on a gold-coated silicon wafer, which was etched to produce free-standing hi layer objects. These bilayers, produced with different thicknesses of Au, were imaged by optical microscopy in suspension in water and in NaCl solutions of varying concentrations. The radius of curvature of these objects was used to extract values for the surface stress induced by the brush in salted and salt-free regimes and to investigate the time scale of the brush swelling transition, demonstrating that this technique can be used to probe the mechanical properties of stimulus-responsive brushes.
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174. Exploiting the superior protein resistance of polymer brushes to control single cell adhesion and polarisation at the micron scale
The control of the cell microenvironment on model patterned substrates allows the systematic study of cell biology in well defined conditions, potentially using automated systems. The extreme protein resistance of poly(oligo(ethylene glycol methacrylate)) (POEGMA) brushes is exploited to achieve high fidelity patterning of single cells. These coatings can be patterned by soft lithography on large areas (a microscope slide) and scale (substrates were typically prepared in batches of 200). The present protocol relies on the adsorption of extra-cellular matrix (ECM) proteins on unprotected areas using simple incubation and washing steps. The stability of POEGMA brushes, as examined via ellipsometry and SPR, is found to be excellent, both during storage and cell culture. The impact of substrate treatment, brush thickness and incubation protocol on ECM deposition, both for ultra-thin gold and glass substrates, is investigated via fluorescence microscopy and AFM. Optimised conditions result in high quality ECM patterns at the micron scale, even on glass substrates, that are suitable for controlling cell spreading and polarisation. These patterns are compatible with state-of-the-art technologies (fluorescence microscopy, FRET) used for live cell imaging. This technology, combined with single cell analysis methods, provides a platform for exploring the mechanisms that regulate cell behaviour. (C) 2010 Elsevier Ltd. All rights reserved.
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173. Controlling nanoscale morphology in polymer photovoltaic devices
Whilst inspiring significant academic interest, the maximum power conversion efficiencies achieved by polymer PVs under solar conditions (PCE; 6.10-6.77%), are not yet sufficient for the devices to become widely marketable. Therefore much current work in the area is focussed on raising device efficiencies as far as possible towards theoretically achievable levels. To this end, key strategies involve material design and synthesis, device processing, and methods for controlling the morphology of the active components. This review aims to highlight the importance of morphological design and control for highly efficient polymer PVs, to discuss strategies by which morphology can be controlled, and to outline some of the characterisation techniques vital to the understanding and optimisation of morphology in these materials. (C) 2010 Elsevier Ltd. All rights reserved.
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172. Poly(9,9-dioctylfluorene)-Based Conjugated Polyelectrolyte: Extended pi-Electron Conjugation Induced by Complexation with a Surfactant Zwitterion
We report on a conjugated polyelectrolyte (CPE) based on fluorene repeat units, which forms a supramolecular complex with a zwitterion surfactant. The complex self-assembles into multilamellar structures on solid substrates. The luminescence efficiency, low in the uncomplexed polymer, is strongly increased after complexation. This originates from the phase segregation between the aromatic backbone and ionic sides, reducing conformational defects and ionic dipole-induced quenching.
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171. Generation of Picoliter Droplets with Defined Contents and Concentration Gradients from the Separation of Chemical Mixtures
There has been an increasing drive toward miniaturizing and accelerating experiments with droplet-based microfluidics across the chemical disciplines. Current applications take advantage of the numerous techniques for manipulating nano- to femtoliter droplets within microfluidic devices. To expand the range of possible applications, we have developed a method for compartmentalizing pure compounds within droplets, at a gradient of concentrations, starting from chemical mixtures. In this technique, a mixture is injected into an ultra performance liquid chromatography (UPLC) system, and droplets are generated from the LC output at a frequency high enough to fraction each compound into similar to 10(5) droplets, compartmentalizing pure compounds into a sequence of droplets with a range of concentrations spanning 2-3 orders of magnitude. Here we used fluorescent dyes to quantify the concentration profile of the droplet collections, and to demonstrate the correspondence between the concentration profile of the droplets and the compound elution profile monitored with a UV absorbance detector, allowing the use of compounds that are not fluorescently labeled but show UV absorbance. Hence this technique is applicable to a wide variety of applications that require both compound purity and the ability to probe a variety of concentrations, such as drug screening and titrations.
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170. Synthesis, Characterization, and Surface Initiated Polymerization of Carbazole Functionalized Isocyanides
We describe the design and synthesis of carbazole functionalized isocyanides and the detailed investigation of their properties. Characterization by solid state NMR, CD, and IR spectroscopic techniques reveals that the polymer has a well-defined helical architecture. Surface-initiated polymerization of the isocyanide monomers onto various surfaces was carried out to give polymer brushes up to 150 nm thick. Insights into the electronic properties of the materials were obtained by Kelvin probe force microscopy (KPFM) and electroabsorption studies
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169. Micron-scale channel formation by the release and bond-back of pre-stressed thin films: A finite element analysis
Buckling of thin films on a rigid substrate during use or fabrication is a well-known but unwanted phenomenon. However, this phenomenon can also be exploited to generate well-controlled patterns at the micro and nano-scale. These patterned surfaces find various technological applications such as optical gratings or micro/nano-fluidic channels. In this article, we present a numerical model that accounts for the buckling-up of pre-strained thin films by a reduction of the interface toughness and the subsequent bond-back. Channels are formed whose dimensions can be controlled by tuning the film dimensions, film thickness and stiffness, the eigenstrain in the film and the cohesive interface energy between the film and the substrate. We will show how the buckling-up and draping back processes can be captured in terms of a limited set of dimensionless parameters, providing quantitative insight on how these parameters should be tuned to generate a specified channel geometry. (C) 2010 Elsevier Ltd. All rights reserved.
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168. Formation of Nanopatterned Polymer Blends in Photovoltaic Devices
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 (10(14)/mm(2)) 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 hererojunctions 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, eta(e)) of 1.9%.
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167. Polymer Brushes: Routes toward Mechanosensitive Surfaces
Soft nanotechnology involves both understanding the behavior of soft matter and using these components to build useful nanoscale structures and devices. However, molecular scale properties such as Brownian motion, diffusion, surface forces, and conformational flexibility dominate the chemistry and physics in soft nanotechnology, and therefore the design rules for generating functional structures from soft, self-assembled materials are still developing. Biological motors illustrate how wet nanoscale machines differ from their macroscopic Counterparts. These molecular machines convert chemical energy into mechanical motion through an isothermal process: chemical reactions generate chemical potential and diffusion of ions, leading to conformational changes in proteins and the production of mechanical force. Because the actuation steps form a thermodynamic cycle that is reversible, the application of mechanical forces can also generate a chemical potential. This reverse process of mechanotransduction is the underlying sensing and signaling mechanism for a wide range of physiological phenomena such as hearing, touch, and growth of bone. Many of the biological systems that respond to mechanical stimuli do this via complex stress-activated ion channels or remodeling of the actin cytoskeleton. These biological actuation and mechanosensing processes are rather different from nano- and microelectromechanical systems (NEMS and MEMS) produced via semiconductor fabrication technologies. In our group, we are working to emulate biological mechanotransduction by systematically developing building blocks based on polymer brushes. In this soft nanotechnology approach to mechanotransduction, the chemical building blocks are polymer chains whose conformational changes and actuation can be investigated at a very basic level in polymer brushes, particularly polyelectrolyte brushes. Because these polymer brushes are easily accessible synthetically with control over parameters such as composition, chain length, and chain density, brushes provide a robust platform to study the coupling of mechanical forces with conformational changes of the chains. This Account provides an overview of our recent research in the design of mechanosensitive polymer brushes starting with the demonstration of nanoactuators and leading to our first attempts toward the creation of artificial mechanotransduction elements. As the brushes collapse in response to external triggers such as pH and ion concentration, polyelectrolyte brushes provide stimuli-responsive films. These collapse transitions lead to the generation of mechanical forces, and by reversing the chain of events, we designed a mechanically responsive film with a chemical output. Having reported an initial proof-of-principle experiment, we think that the stage is set for the preparation of more elaborate mechanosensitive surfaces.
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166. Polyelectrolyte-Bridged Metal/Cotton Hierarchical Structures for Highly Durable Conductive Yarns
A novel, facile, and versatile approach for preparing highly durable, electrically conductive cotton yarns is reported., Polyelectrolyte brushes, a polymer that covalently tethers one end on a surface, are first grown from cotton surfaces by surface-initiated atomic transfer radical polymerization. Subsequent electroless deposition of metal particles onto the brush-modified cotton yarns yields electrically conductive yarns, which have conductivity as high as similar to 1 S/cm and can be used as electrical wires in wearable:, flexible electronic devices. Importantly, the formation of polymer brush-bridged metal/cotton hierarchical structures provides robust mechanical and electrical durability to the yarns under many stretching, bending, rubbing, and washing cycles. With proper selection of metal, the conductivity of the samples remains stable after they are stored in air for a Few months. This chemical approach can be extended as a general method for making conductive yarns and fabrics from all kinds of natural fibers.
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165. Emerging applications of stimuli-responsive polymer materials
Responsive polymer materials can adapt to surrounding environments, regulate transport of ions and molecules, change wettability and adhesion of different species on external stimuli, or convert chemical and biochemical signals into optical, electrical, thermal and mechanical signals, and vice versa. These materials are playing an increasingly important part in a diverse range of applications, such as drug delivery, diagnostics, tissue engineering and ‘smart’ optical systems, as well as biosensors, microelectromechanical systems, coatings and textiles. We review recent advances and challenges in the developments towards applications of stimuli-responsive polymeric materials that are self-assembled from nanostructured building blocks. We also provide a critical outline of emerging developments.
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164. Simultaneous red-green-blue reflection and wavelength tuning from an achiral liquid crystal and a polymer template
A broad and fast wavelength tuning of the photonic bandgap of a hybrid photonic band structure (PBS) formed from an achiral nematic liquid crystal and a polymer template. Remarkably, red-green-blue reflection from a single device and a single achiral nematic liquid crystal mixture is reported without any additional optical components such as a color filter.
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163. Protein-Resistant NTA-Functionalized Polymer Brushes for Selective and Stable Immobilization of Histidine-Tagged Proteins
Protein-resistant polymeric coatings that allow highly selective immobilization of specific biomolecules are essential for biomedical applications such as microarrays, biosensing, heterogeneous catalysis, and bioengineering. Polymer brushes are particularly interesting for this purpose because their chemical structure and physical properties can easily be tailored to meet specific needs. This article explores the functionalization of two protein-resistant polymer brushes, poly(oligoethylene glycol methacrylate) (POEGMA) and poly(hydroxyethyl methacrylate) (PHEMA), with nitrilotriacetic acid (NTA) moieties that can complex histidine-tagged (His-tagged) proteins selectively and reversibly. Using Fluorescence microscopy, IR spectroscopy, X-ray photoelectron spectroscopy, surface plasmon resonanace, and ellipsometry, we demonstrate that His-tagged green fluorescent protein can be immobilized on NTA brushes with high stability and loading. The loading saturation reached for NTA-POEGMA is higher than that for NTA-PHEMA because of increased swelling of the former brush. Despite this higher loading capacity, NTA-POEGMA remained highly protein-resistant, which shows its potential for clean and specific protein immobilization. Finally, we showed that the preserved protein resistance of NTA-POEGMA brushes can be used to generate well-defined binary biofunctional patterns via a simple protocol of incubations and washes. These patterns may find applications in cell arraying and screening.
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162. Microdroplets in Microfluidics: An Evolving Platform for Discoveries in Chemistry and Biology
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161. Synthesis and characterization of low bandgap conjugated donor-acceptor polymers for polymer: PCBM solar cells
We report on the synthesis, characterization and photovoltaic performance of three novel semiconducting polymers based on poly[bis-N,N’-(4-octylphenyl)-bis-N,N’-phenyl-1,4-phenylenediamine-alt-5,5′-4′,7′,-di-2-thienyl-2′,1′,3′-benzothiadiazole]. They differ only in the presence and position of hexyl side-chains on the thienyl groups. T8TBT-0 has no such side-chains, they face towards the benzothiadiazole in T8TBT-in and away in T8TBT-out. Based on electron-donating triarylamine and electron-accepting dithienyl-benzothiadiazole groups, the new polymers exhibit low bandgaps and enhanced absorption in the red part of the visible spectrum. Despite their identical backbone they differ in their synthesis and photophysics: T8TBT-0 and T8TBT-in can be synthesized by direct Suzuki coupling but a new synthesis procedure is necessary for T8TBT-out. In absorption and luminescence a blue shift is induced by the inward facing, and to a lesser extent by the outward-facing side-chains. From comparison of the photophysics in solutions and films, we conclude that the addition of side-chains reduces formation of aggregates in films and that this effect is stronger for inward-facing side-chains. By blending the three polymers with PCBM in a standard photovoltaic device architecture, T8TBT-0 performs best with a power conversion efficiency (PCE) of 1.0% (under AM1.5G illumination at 100 mW cm(-2)) compared to 0.17% and 0.27% for T8TBT-out and T8TBT-in, respectively.
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160. A double droplet trap system for studying mass transport across a droplet-droplet interface
Here we present the design, fabrication and operation of a microfluidic device to trap droplets in a large array of droplet pairs in a controlled manner with the aim of studying the transport of small molecules across the resultant surfactant bilayers formed between the droplet pairs.
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159. Hydrophilic PDMS microchannels for high-throughput formation of oil-in-water microdroplets and water-in-oil-in-water double emulsions
Here we present a novel surface modification method based on the sequential layer-by-layer deposition of polyelectrolytes yielding hydrophilic microchannels in PDMS-based microfluidic devices. The coatings are long-term stable and allow for the generation of monodisperse oil-in-water microdroplets even several months after the channel surface treatment. Due to the robustness of the polyelectrolyte multilayers ultra-high flow rates can be applied, making high-throughput droplet formation in the jetting mode possible. Furthermore, we successfully used our method to selectively modify the surface properties in certain areas of assembled microchannels. The resulting partially hydrophilic, partially hydrophobic microfluidic devices allow for the production of monodisperse water-in-oil-in-water double emulsions.
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158. Simultaneous Determination of Gene Expression and Enzymatic Activity in Individual Bacterial Cells in Microdroplet Compartments
A microfluidic device capable of storing picoliter droplets containing single bacteria at constant volumes has been fabricated in PDMS. Once captured in droplets that remain static in the device, bacteria express both a red fluorescent protein (mRFP1) and the enzyme, alkaline phosphatase (AP), from a biscistronic construct. By measuring the fluorescence intensity of both the mRFP1 inside the cells and a fluorescent product formed as a result of the enzymatic activity outside the cells, gene expression and enzymatic activity can be simultaneously and continuously monitored. By collecting data from many individual cells, the distribution of activities in a cell is quantified and the difference in activity between two AP mutants is measured.
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157. Electrically Tuneable Liquid Crystal Photonic Bandgaps
Electrically controlled color tuning of the photonic bandgap and electrically induced multiple photonic bandgaps from a chiral nematic liquid crystal mixture doped with a ferroelectric liquid crystal.
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156. Biofunctionalized Protein Resistant Oligo(ethylene glycol)-Derived Polymer Brushes as Selective Immobilization and Sensing Platforms
Poly(oligo(ethylene glycol) methacrylate) (POEGMA) brushes are extremely protein resistant polymer coatings that can reduce nonspecific adsorption of proteins from complex mixtures such as blood, sera and plasma. These coatings can be prepared via atom transfer radical polymerization with excellent control of their thickness and grafting density. We studied their direct functionalization with streptavidin and developed an assay for determining which coupling conditions afford the highest streptavidin loading efficiency. Disuccinimidyl carbonate was found to be the most efficient activating agent for covalent capture of the receptor. Using infrared and X-ray photoelectron spectroscopy, fluorescence microscopy, surface plasmon resonance, and ellipsometry, we examined how structural parameters such as the length of the oligo(ethylene glycol) side chain affect streptavidin functionalization, but also immobilization of biotinylated antibodies, subsequent selective secondary recognition and nonspecific binding of proteins. We found evidence that large macromolecules cannot infiltrate dense polymer brushes and that bulky antibody recognition occurs in the upper part of these coatings.
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155. Formation of Hierarchically Structured Thin Films
Here, we report the preparation of hierarchically structured polymer brushes with well-defined geometries via multiple step microcontact printing (MS-mu CP) of inks containing different ratios of initiator-terminated thiols and non-reactive alkylthiols. Thick (and dense), polymer brushes grew from self-assembled monolayers (SAMs) with high concentration of initiator-terminated thiols, and these brushes exhibited high chemical etch-resistance, compared to thin (and less dense), brushes grown from more dilute initiator-terminated SAMs. Upon etching, patterned crosslinking polymer brush films decorated with thin layers of Au, could be lifted off the surface to form geometrically well-defined free-standing hierarchical films. These polymer brush films showed interesting buckling instabilities when compressed. Areas with different brush thicknesses and Au backing showed markedly different buckling behavior, leading to unusual patterns of wrinkles with different wavelengths and orientations toward the force field.
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154. Ion-Induced Formation of Charge-Transfer States in Conjugated Polyelectrolytes
We use time-resolved optical spectroscopy to demonstrate that the luminescence quenching observed when ions are incorporated in films of conjugated polymers can be explained by the formation of charge-transfer (CT) states that are stabilized by the Coulomb field of ions. Our investigation is focused on a conjugated polyelectrolyte (CPE) derived from F8BT (poly(9,9′-dioctylfluorene-a/t-benzothiadiazole)). The statistical copolymer contains tetra-alkyl ammonium moieties and BF4- counteranions attached to a moderate (similar to 7%) density of polymer alkyl side chains, providing a film morphology comparable to F8BT but with ions distributed on the length scale of exciton diffusion. The ionic substituents have little influence over the polymers electronic absorption and emission properties in solution, however photoluminescence (PL) quantum efficiency (similar to 6%) is considerably lower for the polyelectrolyte compared with F8BT (similar to 60%) in thin films. Time-resolved PL spectroscopy reveals that the primary exciton lifetime is shortened in the polyelectrolyte and a red-shifted CT emission peak with a longer lifetime emerges. Transient absorption spectroscopy of thin films enables us to detect CT states that persist beyond the primary decay and are found to be immobile. The PL intensity of the partially ionic film is found to increase with decreasing temperature, consistent with thermally activated exciton hopping (E-act = 28 meV) prior to formation of CT states at ionic regions. We suggest that ion-induced stabilization of CT states is a general phenomenon in CPEs, which raises the possibility that ions might be arranged to direct the flow of excitons toward charge-separating interfaces in polymer photovoltaic devices.
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153. Nanopatterning via Pressure-Induced Instabilities in Thin Polymer Films
The residual stresses in spin-coated films can be exploited to produce highly controlled nanoscale patterns via pressure-induced local rupturing and dewetting of thin films. Residue-free holes as small as 28 nm in diameter formed over large areas by pressing sharp stamps into polymer films at temperatures well below the glass transition temperature.
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152. Controlling the Retention of Small Molecules in Emulsion Microdroplets for Use in Cell-Based Assays
Water-in-oil microdroplets in microfluidics are well-defined individual picoliter reaction compartments and, as such, have great potential for quantitative high-throughput biological screening. This, however, depends upon contents of the droplets not leaking out into the oil phase. To assess the mechanism of possible leaking, the retention of various fluorescein derivatives from droplets formed in mineral oil and stored for hours in a reservoir on chip was studied. Leaking into the oil phase was observed and was shown to be dependent on the nature of the compounds and on the concentration of the silicone-based polymeric surfactant Abil EM 90 used. In experiments in which droplets filled with fluorescein were mixed with droplets filled with only buffer, the rate of efflux from filled droplets to empty droplets was dependent on the number of neighboring droplets of different composition. Buffer droplets with five fluorescein-containing neighbors took up the fluorophore 4.5 times faster than buffer droplets without fluorescein neighbors. The addition of bovine serum albumin (BSA) substantially reduced leaking. A formulation with 5% BSA reduces leaking of the fluorophore from 45% to 3%. Inclusion of BSA enabled experiments to be carried out over periods up to 18 h, without substantial leaking (<5%. We demonstrate the utility of this additive by following the enzymatic activity of alkaline phosphatase expressed by Escherichia coli cells. The ability to reliably compartmentalize genotype (cell) and phenotype (reaction product) is the basis for using microdroplets; in directed evolution studies, and the approaches described herein provide a test system for assessing emulsion formulations for such purposes.
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151. Direct Measurement of Normal and Shear Forces between Surface-Grown Polyelectrolyte Layers
This paper presents measurements, using the surface force balance (SFB), of the normal and shear forces in aqueous solutions between polyelectrolyte layers grown directly on mica substrates (grafted-from). The grafting-from was via surface-initiated atom transfer radical polymerization (surface-initiated ATRP) using a positively charged methacrylate monomer. X-ray reflectometry measurements confirm the successful formation of polyelectrolyte layers by this method. Surface-inititated ATRP has the advantages that the polymer chains can be strongly grafted to the substrate, and that high grafting densities should be achievable. Measured normal forces in water showed a long-range repulsion arising from an electrical double layer that extended beyond the polyelectrolyte layers, and a stronger, shorter-range repulsion when the polyelectrolyte brushes were in contact. Swollen layer thicknesses were in the range 15-40 nm. Upon addition of similar to 10(-2)-10(-1) M sodium nitrate, screening effects reduced the electrical double layer force to an undetectable level. Shear force measurements in pure water were performed, and the measured friction may arise from polymer chains bridging between the surfaces.
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149. Nonadhesive polymer brush-nanocoatings modified by a natural antimicrobial peptide
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148. The effects of surface chemistry on epidermal stem cell differentiation
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147. Bacterial and mammalian cell response to poly(3-sulfopropyl methacrylate) brushes loaded with silver halide salts
This Study investigates the antibacterial and cytotoxic effect Of Surfaces with sulphonate brushes containing silver salts. By using the same type of samples for both cytotoxicity and antibacterial Studies, these two parameters Could be compared in a controlled way. The silver was incorporated into the brush in four different forms to enable release of silver ions at different concentrations and different rates. It was found that although the Surfaces displayed very good antibacterial properties in buffer Solutions, this effect disappeared in systems with high protein content. Similarly, the silver-containing Surfaces displayed cytotoxic effects in the absence of serum proteins but this effect was reduced in the presence of serum. The speciation of silver in the different Solutions is discussed. Cytotoxic and antibacterial effects are compared at the different silver concentrations released. The implications of a concentration range where silver could be used to kill bacterial Without harmful effects on mammalian cells are also discussed and questioned. (C) 2008 Elsevier Ltd. All rights reserved.
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146. Density control of ZnO nanowires grown using Au-PMMA nanoparticles and their growth behavior
Au nanoparticles stabilized by poly(methyl methacrylate) (PMMA) were used as a catalyst to grow vertically aligned ZnO nanowires (NWs). The density of ZnO NWs with very uniform diameter was controlled by changing the concentration of Au-PMMA nanoparticles (NPs). The density was in direct proportion to the concentration of Au-PMMA NPs. Furthermore, the growth process of ZnO NWs using Au-PMMA NPs was systematically investigated through comparison with that using Au thin film as a catalyst. Au-PMMA NPs induced polyhedral-shaped bases of ZnO NWs separated from each other, while Au thin film formed a continuous network of bases of ZnO NWs. This approach provides a facile and cost-effective catalyst density control method, allowing us to grow high-quality vertically aligned ZnO NWs suitable for many viable applications.
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145. Organization of Nanoparticles in Polymer Brushes
We have demonstrated a facile infiltration process, in which gold nanoparticles are assembled into block copolymer brushes. After solvent annealing, the polymer-covered nanoparticles are either sequestered into the corresponding block copolymer domain or expulsed from the brush, depending on the shell density of the nanoparticles.
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144. Photoresponsive Polymer Brushes for Hydrophilic Patterning
The use of photolabile protecting groups (PGs) as a means to create latent hydrophilic surfaces is presented. Naturally hydrophobic PGs, based on o-nitrobenzyl chemistry, are used on polymer side chains, poised for cleavage upon exposure to UV light. Removal of the PGs liberates the hydrophilic polymer, thereby switching the surface wettability from hydrophobic to hydrophilic. This switch can be augmented by increasing the surface roughness. Additionally, this system is also shown to be spatially addressable, a highly desirable property for applications which require specific regions of a surface to switch their wettability.
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143. Coupling Microdroplet Microreactors with Mass Spectrometry: Reading the Contents of Single Droplets Online
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142. Antibacterial and Antifouling Polymer Brushes Incorporating Antimicrobial Peptide
Surface-initiated atom transfer radical polymerization (ATRP) has been used to prepare antifouling copolymer brushes based on 2-(2-methoxyethoxy)ethyl methacrylate (MEO(2)MA) and hydroxyl-terminated oligo(ethylene glycol) methacrylate (HOEGMA). The amount of hydroxyl reactive groups incorporated into the brushes was varied by changing the composition of the monomer mixture. These coatings were subsequently functionalized by a natural antibacterial peptide, magainin I, via an oriented chemical grafting on hydroxyl groups, which maintains the activity of the peptide. The antibacterial activity of the functionalized brushes was successfully tested against two different strains of gram-positive bacteria.
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141. Suzuki-Miyaura coupling reactions in aqueous microdroplets with catalytically active fluorous interfaces
Using microfluidic techniques and a novel fluorous-tagged palladium catalyst, we generated droplet reactors with catalytically active walls and used these compartments for small molecule synthesis.
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140. Electrolyte-modulated electrochemistry and electrocatalysis on ferrocene-terminated polyelectrolyte brushes
We prepared a responsive polyelectrolyte (ferrocene-terminated poly[1-ethyl 3-(2-methacryloyloxyethyl) imidazolium chloride], PEMEImCl) brush on an Au surface through a three-step procedure consisting of surface-initiated atom transfer radical polymerization, followed by treatment with NaN3 and by a reaction with ethynylferrocene via azide-alkyne Huisgen cycloaddition reaction, known as ‘click’ chemistry. The interfacial resistance of the polyelectrolyte brush was characterized with electrochemical impedance spectroscopy (EIS) using [Fe(CN)(6)](3-/4-) as the redox probe in different electrolyte solution. Moreover, the redox behavior of ferrocene (Fc) at the terminus of polyelectrolyte brushes in different electrolyte solutions was characterized by cyclic voltammetry (CV). The electrochemical properties depended on the distance between redox probes (Fc at the terminus) and the substrate, which were affected by the conformation of the polyelectrolyte brush triggered by external environments. The electrocatalysis behavior of the Fc-terminated polyelectrolyte brush also can be modulated via the conformation of the brush. It is interesting that all the electrochemical behavior of the polyelectrolyte brush can be mediated by the kind and concentration of anion.
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139. Simultaneous measurement of reactions in microdroplets filled by concentration gradients
This work describes a technology for performing and monitoring simultaneously several reactions confined in strings of microdroplets having identical volumes but different composition, and travelling with the same speed in parallel channels of a microfluidic device. This technology, called parallel microdroplets technology (P mu D), uses an inverted optical microscope and a charge-coupled device (CCD) camera to collect images and analyze them so as to report on the reactions occurring in these microdroplets. A concentration gradient of one reactant is created in the microfluidic device. In each channel, a different concentration of this reactant is mixed with a fixed amount of a second reactant. Using planar flow-focusing methodology, these mixtures are confined in microdroplets of pL size which travel in oil as continuous medium, avoiding laminar dispersion. By analyzing the images of parallel strings of microdroplets, the time courses of several reactions with different reagent compositions are investigated simultaneously. In order to design the microfluidic device that consists in a complex network of channels having well-defined geometries and restricted positions, the theoretical concept of equivalent channels (i.e. channels having identical hydraulic resistance) is exploited and developed. As a demonstration of the P mu D technology, an enzyme activity assay was carried out and the steady-state kinetic constants were determined.
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138. The switching properties of chiral nematic liquid crystals using electrically commanded surfaces
In this paper, we describe in detail the electrical photonic band gap (PBG) tuning properties of a chiral nematic liquid crystal (N*LCs) using electrically active ferroelectric liquid crystals (FLCs) to generate surface mediated switching. The tuning of the PBG occurs as a result of a contraction of the helix which is induced passively by the in-plane rotations of the N*LC molecules at the FLC/N*LC interface. We provide detail on the preparation of the samples and discuss how the tuning range is altered when the number of switching surfaces increases from just one surface to both top and bottom surfaces. The effect is found to be completely reversible and there is no apparent change in the quality of the structure during tuning. It is observed, however, that the time required to reach equilibrium once an electric field is applied or removed is much longer than the response time of the FLC confined between conventional polyimide rubbed substrates. This is due to the elastic restoring force and viscosity of the N*LC. Nevertheless, this approach to tuning the band gap is of potential use in biomedicine and sensing applications whereby a tunable band-pass filter and/or tunable lasers are required.
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137. Surface modification of PDMS via self-organization of vinyl-terminated small molecules
Polydimethylsiloxane (PDMS) elastomers are widely used in soft lithography, microfluidics and biomedical applications as they combine a range of desirable chemical and physical properties. We studied the surface of PDMS modified using surface enrichment with long-chain alkenes. These alkenes were immobilized during crosslinking of the PDMS pre-polymer mixture cast against a template of matching surface energy. A range of different functional groups was introduced, including perfluorinated and oligoethylene glycol groups, alkyl chains and initiators for controlled radical polymerization. A detailed analysis of the composition of the modified PDMS surfaces was carried out using contact angle measurements, AFM, SIMS and XPS. The results demonstrate that the PDMS was enriched with small molecules near the surface and show that the functional molecules follow the template surface energy on patterned surfaces with edge resolutions equal to the template. By introducing hydrophilic alkenes receding contact angles on PDMS can be lowered below 5 degrees, and we investigated how these surfaces rearrange in air due to the chain mobility of the PDMS backbone and PDMS short-chain fragments.
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136. Polymer brush resist for responsive wettability
Well-defined polymer films which change their wettability in response to external stimuli are extensively used within the microelectronics industry, and are rapidly becoming equally beneficial in the field of microfluidics and lab-on-a-chip technologies. The fabrication of a self-contained polymer brush resist, by using the chemistry of surface initiated atom transfer radical polymerization (SI-ATRP) and photoacid generators (PAGs), and its subsequent responsive wettability will be the subject of this paper. Since UV light is used to trigger the responsive behaviour, spatially addressable patterning could be achieved, without the use of sacrificial layers, allowing for the formation of single films with regions of different wettabilities.
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135. Collective Behavior of Magnetic Nanoparticles in Polyelectrolyte Brushes
Polyelectrolyte brushes loaded with magnetic nanoparticles can be formed through the reaction of immobilized Fe2+/3+, ions in the brush. These particles show very unusual magnetic properties, not normally associated with such small nanoparticles. Upon application of all external magnetic field, the brushes showed reversible dimensional changes.
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134. Direct Visualization of Reversible Switching of Micropatterned Polyelectrolyte Brushes on Gold Surfaces Using Laser Scanning Confocal Microscopy
We apply confocal fluorescence microscopy for real time studies of reversible conformational changes of poly(methacryloyloxyethyl phosphate) (PMEP) brushes chemically grafted onto gold substrates. Oregon green 488 fluorophores chemically attached onto the PMEP polymers were used as reporters for probing the conformational changes. Use of a specially designed liquid flow microchamber allowed dynamic imaging of the brushes under varying environmental conditions. The fluorescence intensities exhibited fully reversible brightness changes on alternation of the solution in the chamber between water and KCl. This reversible quenching behavior is consistent with a conformational change between an extended and a collapsed brush configuration. The fluorescence quenching behavior of the brushes was found to be dependent on ion concentration as well as polymer grafting density and was caused by nonradiative energy transfer to the polymer scaffold and the gold substrate.
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133. Thickness-Dependent Properties of Polyzwitterionic Brushes (vol 41, pg 6317, 2008)
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132. Effect of Nanoconfinement on the Collapse Transition of Responsive Polymer Brushes
Nanopatterned brushes of a thermo-responsive polymer, poly(2-(2-methoxyethoxy)ethyl methacrylate) (PMEO(2)MA), displaying a collapse temperature in the physiological range were synthesized for grafting diameters from a few micrometers down to 35 nm. The reversible collapse transition of the nanobrushes was studied in water as a function of their lateral confinement, down to ensembles of brushes containing only similar to 300 chains. The confinement results in a considerable broadening of the collapse transition and in an increase of the degree of vertical swelling, which can be explained by the internal structure of the nanodroplets derived from a theoretical model of dry nanobrushes. These results enable the rational design of responsive surfaces having a tunable topography engineered at the nanometer scale, which is of direct interest for the development of soft nanoactuators and new substrates for cell adhesion studies.
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131. Chain entropy and wetting energy control the shape of nanopatterned polymer brushes
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130. Surface-Directed Phase Separation of Conjugated Polymer Blends for Efficient Light-Emitting Diodes
The ability to control organic-organic interfaces in conjugated polymer blends is critical for further device improvement. Here, we control the phase separation in blends of poly (9,9-di-n-octylfluorene-alt-benzothiadiazole) (F8BT) and poly(9,9-di-n-octylfluorene-alt-(1,4-phenylene-((4-sec-butylphenyl)imino)-1,4-phenylene) (TFB) via chemical modification of the substrate by microcontact printing of octenyltrichlorosilane molecules. The lateral phase-separated structures in the blend film closely replicate the underlying micrometer-scale chemical pattern. We found nanometer-scale vertical segregation of the polymers within both lateral domains, with regions closer to the substrate being substantially pure phases of either polymer. Such phase separation has important implications for the performance of light-emitting diodes fabricated using these patterned blend films. In the absence of a continuous TFB wetting layer at the substrate interface, as typically formed in spin-coated blend films, charge carrier injection is confined in the well-defined TFB-rich domains. This confinement leads to high electroluminescence efficiency, whereas the overall reduction in the roughness of the patterned blend film results in slower decay of device efficiency at high voltages. In addition, the amount of surface out-coupling of light in the forward direction observed in these blend devices is found to be strongly correlated to the distribution of periodicity of the phase-separated structures in the active layer.
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129. Reversible electrochemical switching of polyelectrolyte brush surface energy using electroactive counterions
Polyelectrolyte brushes with electroactive counterions provide an effective platform for surfaces with electrochemically switchable wetting properties. Polycationic poly(2-(methacryloyloxy)-ethyl-trimethyl-ammonium chloride) (PMETAC) brushes with ferricyanide ions ([Fe(CN)(6)](3-)) were used as the electrochemically addressable surface. After a negative potential of -0.5 V was applied to the [Fe(CN)(6)](3-)-coordinated PMETAC brushes, the [Fe(CN)(6)](3-) species were reduced to [Fe(CN)(6)](4-), and the surface became more hydrophilic. By application of alternating negative and positive potentials, PMETAC brushes were switched reversibly between the reduced state ([Fe(CN)6]4-) and oxidized state ([Fe(CN)(6)](3-)), resulting in reversible changes in water contact angles. The time required for a complete contact angle change can be tuned from 1 to 20 s, by changing the brush thickness and the concentration of supporting electrolyte. We present an electrochemical brush transport model that includes the electrochemical reaction at the charged electrode and describes ion transport through the brush phase covering the electrode. The model quantitatively describes the response of the contact angle (hydrophilicity) to the applied voltage as a function of background ionic strength and brush thickness, supporting the proposed mechanism of ion transport through the brush and electrochemical reaction at the electrode. A typical diffusion constant for ferricyanide in a PMETAC brush of any thickness in 5 mM KCl supporting electrolyte was found to be 2 x 10(-15) m(2) s(-1), 5 to 6 orders of magnitude smaller than its bulk solution value.
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128. Thickness-dependent properties of polyzwitterionic brushes
This paper describes detailed studies on the controlled synthesis of poly [2-(methacryloyloxy)ethyl]dimethyl(3-sulfopropyl)ammonium hydroxide (polyMEDSAH) from initiator-modified gold surfaces and the properties of these brushes as a function of grafting density and thickness. Improved control over polymerization was achieved by preforming the catalytically active complex to ensure that the monomers did not coordinate to the catalyst. We observed an intriguing transition thickness (h(crit)) in which the polyMEDSAH brushes switched from hydrophilic to hydrophobic due to the strong inter- and intrachain associations. We studied this transition as a function of the rate of polymerization and grafting density and found that both factors strongly influence the value of h(crit). Faster grown polyMEDSAH brushes have higher h(crit) values, and brushes grown from higher graffing density have higher h(crit) value. These observations suggest that the h(crit) is governed by the density of the brushes, the polymer chain length, and the degree of alignment. Furthermore, the inter- or intrachain associations can be reversed by increasing the temperature, leading to a hydrophobic to hydrophilic switch, where the magnitude of the switching is governed by the grafting density.
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127. ANYL 24-Selective emulsion separation toward integration of microdroplets with microfluidic analytical techniques
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126. PMSE 163-Polyelectrolyte brushes as nanoscale actuators
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125. PMSE 90-Controlled infiltration of gold nanoparticles into polymer brushes
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124. Responsive polymers for nanoscale actuation
Soft nanotechnology is a rapidly developing area of research that exploits principles such as self-assembly, entropy, swelling and collapse transitions, and polymeric building blocks to emulate actuation principles observed in natural systems. Unlike lithographically fabricated devices, soft nanotechnology uses much less regularly structured and largely organic materials, deriving their energy from chemical reactions and with macroscopic functionality arising from nanoscale conformational changes. In this review, some recent developments in nanostructured polymer gels and polymer brushes are discussed, which provide promising new directions for exploiting soft materials as nanoactuators.
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123. Development of quantitative cell-based enzyme assays in microdroplets
We describe the development of an enzyme assay inside picoliter microdroplets. The enzyme alkaline phosphatase is expressed in Escherichia coli cells and presented in the periplasm. Droplets act as discrete reactors which retain and localize any reaction product. The catalytic turnover of the substrate is measured in individual droplets by monitoring the fluorescence at several time points within the device and exhibits kinetic behavior similar to that observed in bulk solution. Studies on wild type and a mutant enzyme successfully demonstrated the feasibility of using microfluidic droplets to provide time-resolved kinetic measurements.
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122. Efficient conjugated-polymer optoelectronic devices fabricated by thin-film transfer-printing technique
The fabrication of functional multilayered conjugated-polymer structures with well-defined organic-organic interfaces for optoelectronic-device applications is constrained by the common solubility of many polymers in most organic solvents. Here, we report a simple, low-cost, large-area transfer-printing technique for the deposition and patterning of conjugated-polymer thin films. This method utilises a planar poly(dimethylsiloxane) (PDMS) stamp, along with a water-soluble sacrificial layer, to pick up an organic thin film (similar to 20 nm to 1 mu m) from a substrate and subsequently deliver this film to a target substrate. We demonstrate the versatility of this transfer-printing technique and its applicability to optoelectronic devices by fabricating bilayer structures of poly(9,9-di-n-octylfluorene-alt-(1,4-phenylene-((4-sec-butylphenyl)imino)-1,4-phenylene))/poly(9,9-di-n-octylfluorene-alt-benzo-thiadiazole) (TFB/F8BT) and poly(3-hexylthiophene)/methanofullerene([6,6]-phenyl C-61 butyric acid methyl ester) (P3HT/ PCBM), and incorporating them into light-emitting diodes (LEDs) and photovoltaic (PV) cells, respectively. For both types of device, bilayer devices fabricated with this transfer-printing technique show equal, if not superior, performance to either blend devices or bilayer devices fabricated by other techniques. This indicates well-controlled organic-organic interfaces achieved by the transfer-printing technique. Furthermore, this transfer-printing technique allows us to study the nature of the excited states and the transport of charge carriers across well-defined organic interfaces, which are of great importance to organic electronics.
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121. PMSE 26-Chemical micropatterning of poly(dimethylsiloxane) by self-assembly of functional molecules at the interface
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120. Exploring actuation and mechanotransduction properties of polymer brushes
Soft nanotechnology requires new approaches and materials to efficiently convert chemical energy into mechanical motion and vice versa. A number of key design parameters, such as responsiveness to external stimuli, directionality of response through alignment, transduction via surface stresses or changes in ionic conductivity can be found in polymer brushes and several recent examples of actuation and transduction in polymer brushes will be explored.
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119. Synthesis and characterization of surface-initiated helical polyisocyanopeptide brushes
This article describes the first surface-initiated polymerization of isocyanide monomers onto various surfaces in a controlled manner. Brushes up to 200 nm could be easily obtained within 3 h of polymerization, and the polymer growth was studied as a function of reaction time, monomer concentration, and growth conditions, using atomic force microscopy (AFM) and ellipsometry. The monomers used in this study contain dipeptide (L,L-Ala-Ala) side groups; circular dichroism (CD) measurements, Fourier transform infrared spectroscopy (FTIR), and AFM confirmed that the well-defined helical conformation was retained in the polyisocyanide brushes with hydrogen bonds along the polymer chains.
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118. Polarization anisotropy dynamics for thin films of a conjugated polymer aligned by nanoimprinting
Time-integrated and femtosecond time-resolved photoluminescence spectroscopy has been used to study the dynamic emission polarization anisotropy for thin films of a conjugated polymer whose chains had been aligned through a nanoimprinting technique. The results indicate a high degree of chain alignment, with the presence of a small fraction of unaligned chain domains in film regions far from the imprinted surface. The time-averaged emission from aligned domains is found to be slightly shifted to higher photon energies compared to that from more disordered film regions. This effect is attributed to a subtly different chain packing geometry in the more aligned regions of the film, which leads to a reduced exciton diffusivity and inhibits energetic relaxation of the exciton in the inhomogeneously broadened density of states. While for an unaligned reference film, exciton migration results in a nearly complete depolarization of the emission over the first 300 ps, for the aligned films, interchain exciton hopping from unaligned to aligned domains is found to increase the anisotropy over the same time scale. In addition, excitons generated in aligned film domains were found to be slightly more susceptible to nonradiative quenching effects than those in disordered regions deeper inside the film, suggesting a marginally higher defect density near the nanoimprinted surface of the aligned film.
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117. Polyelectrolyte brush amplified electroactuation of microcantilevers
This paper describes the electroactuation of microcantilevers coated on one side with cationic polyelectrolyte brushes. We observed very strong cantilever deflection by alternating the potential on the cantilever between +0.5 and -0.5 V at frequencies up to 0.25 Hz. The actuation resulted from significant increases in the expansive stresses in the polymer brush layer at both negative and positive potentials. However, the deflection at negative bias was significantly larger. We have developed a theoretical framework that correlates conformational changes of the polymer chains in the brush layer with the reorganization of ions due to the potential bias. The model predicts a strong increase in the polymer volume fraction, close to the interface, which results in large expansive stresses that bend the cantilever at negative potentials. The model also predicts that the actuation responds much stronger to negative potentials than positive potentials, as observed in the experiments.
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116. An integrated device for monitoring time-dependent in vitro expression from single genes in picolitre droplets
Microdroplets have great potential for high-throughput biochemical screening. We report the design of an integrated microfluidic device for droplet formation, incubation and screening. Picolitre water-in-oil droplets can be stored in a reservoir that contains 106 droplets. In this reservoir droplets are stable for at least 6 h, which gives an extended timescale for biochemical experiments. We demonstrate the utility of the system by following the in vitro expression of green fluorescent protein. The high efficiency allows protein expression from a single molecule of DNA template, creating monoclonal droplets in which genotype and phenotype are combined in one emulsion compartment.
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115. Organic thin film transistors with polymer brush gate dielectrics synthesized by atom transfer radical polymerization
Low operating voltage is an important requirement that must be met for industrial adoption of organic field-effect transistors (OFETs). We report here solution fabricated polymer brush gate insulators with good uniformity, low surface roughness and high capacitance. These ultra thin polymer films, synthesized by atom transfer radical polymerization (ATRP), were used to fabricate low voltage OFETs with both evaporated pentacene and solution deposited poly(3-hexylthiophene). The semiconductor-dielectric interfaces in these systems were studied with a variety of methods including scanning force microscopy, grazing incidence X-ray diffraction and neutron reflectometry. These studies highlighted key differences between the surfaces of brush and spun cast polymethyl methacrylate (PMMA) films.
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114. From microdroplets to microfluidics: Selective emulsion separation in microfluidic devices
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113. Localized surface plasmon resonance (LSPR) sensitivity of Au nanodot patterns to probe solvation effects in polyelectrolyte brushes
We report localized surface plasmon resonance (LSPR) of hexagonal Au nanodot arrays to detect changes in the thickness and conformation of poly[ 2-(methacryloyloxy)ethyltrimethyl ammonium chloride] (PMETAC) brushes.
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112. The electrochemical detection of droplets in microfluidic devices
This paper presents a new electrochemical method for the detection and characterisation of aqueous droplets in an organic carrier fluid ( 1,2-dichloroethane) formed in flow-focusing microfluidic devices. The devices consist of a conventional flow-focusing channel 250 mu m wide and 250 mu m deep cast out of poly( dimethylsiloxane) ( PDMS) which is sealed onto a glass substrate containing a set of microelectrodes 100 mu m long. Chronoamperometric analysis of a suitable electrolyte contained in the organic phase is presented for characterising the droplet frequency and size. This chronoamperometric method is then extended to a dual working electrode approach in order to determine the velocity of the droplet. Good agreement between experimental measurements and theory was observed.
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111. Wavelength tuning the photonic band gap in chiral nematic liquid crystals using electrically commanded surfaces
In this letter, the authors demonstrate photonic band gap (PBG) tuning in chiral nematic liquid crystals using electrically commanded surfaces consisting of a ferroelectric liquid crystal. The electrically commanded surfaces generate a contraction of the pitch of the chiral nematic which is manifested as a blueshift of the PBG. Results are presented which demonstrate that tuning can be achieved by either varying the frequency or the amplitude of the electric field. The maximum shift observed for the long-wavelength band edge was 23 nm for an electric field strength of 20 V mu m(-1) and a frequency of 800 Hz to 1 kHz.
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110. Electrochemical characteristics of polyelectrolyte brushes with electroactive counterions
Cyclic voltammetry (CV) was employed to characterize the electrochemical behavior of polyelectrolyte brushes with immobilized electroactive counterions in response to external changes in concentration and composition of the supporting electrolyte and as a function of brush thickness. Poly(methacryloyloxy)ethyl-trimethyl-ammonium chloride (PMETAC) brushes were synthesized on An substrates via atom transfer radical polymerization followed by ion-exchange with ferricyanide, ions ([Fe(CN)(6)](3-)) as redox probes. CV measurements of the modified PMETAC brushes showed the typical electrochemical response corresponding to a surface-confined electroactive species and the redox counterions, as [Fe(CN)(6)](3-) species form stable ion pairs with the quaternary ammonium groups of the brush. The electron-transfer features of PMETAC brushes with different thicknesses, as characterized by CV and UV-vis spectroscopy, revealed that the charge density probed by CV was lower than the charge density measured by UV-vis spectroscopy. The electrode current decreased significantly with increasing concentration of supporting electrolyte due to the effect of the Donnan potential. Hydrophobic counterions, ClO4-, which induced brush collapse, lead to significantly reduced electrode currents.
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109. Synthesis of gold nanoparticles inside polyelectrolyte brushes
In this work we report on the synthesis and characterization of Au nanoparticles grown in the inner environment of cationic polyelectrolyte brushes. The nanocomposite synthesis relies on loading the macromolecular film with AuCl4_ Precursor ions followed by their in situ reduction to Au nanoparticles. We observed that the nanoparticles are uniform in size and are fully stabilized by the surrounding polyelectrolyte chains. Moreover, XRR analysis revealed that the An NPs are formed within the polymer-brush layer. AFM experiments confirmed that the swelling behaviour of the brush layer is not perturbed by the presence of the loaded NPs. The An NP-poly-METAC nanocomposite is remarkably stable to aqueous environments, suggesting the feasibility of using this kind of nanocomposite systems as robust and reliable stimuli-responsive platforms.
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108. Crosslinking polymer brushes for the fabrication of quasi-2-D micro-objects
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107. Synthesis of supercritical carbon dioxide soluble perfluorinated dendrons for surface modification
The rational design, synthesis, and characterization of a series of novel perfluorinated dendrons 14a,b, 25a,b, 26a,b, and 18 are described. The dendrimers were designed to have a thiol at the focal point for attachment to a gold surface to enable the fabrication of self-assembled monolayers (SAMs). Perfluorinated tails were attached to the periphery to provide solubility in supercritical carbon dioxide, and to increase the hydrophobicity and the stability of self-assembled monolayers formed. Mitsunobu reactions were utilized to provide high-yielding steps allowing large-scale production of the novel dendrimers.
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106. Explanation for the apparent absence of collapse of polyelectrolyte brushes in the presence of bulky ions
The conformational behavior of poly[2-(methacryloyloxy)ethyltrimethylammonium chloride] (PMETAC) brushes with different chain density in the presence of large benzyltributylammonium chloride (BTBAC) ions has been studied by a Quartz Crystal Microbalance with Dissipation (QCM-D) and Scanning Force Microscopy. Dense brushes do not collapse in the presence of BTBAC solutions of increasing ionic strength, contrary to what is observed in the presence of NaCl. Brush collapse can be observed for low-ionic-strength solutions of BTBAC when the brush density has been reduced. These phenomena can be explained by considering the Hofmeister series as well as ion size and free space in the brush.
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105. Thermo-responsive polymer brushes with tunable collapse temperatures in the physiological range
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104. Uniaxial alignment of liquid-crystalline conjugated polymers by nanoconfinement
We demonstrate the uniaxial alignment of a liquid-crystalline conjugated polymer, poly(9,9-dioctylfluorene-co-benzothiadiazole) (F8BT) by means of nanoconfinement during nanoimprinting. The orientation of the conjugated backbones was parallel to the nanolines imprinted into the polymer film. Polarized UV-vis absorption and photoluminescence spectra were measured to quantify the degree of alignment, showing that the polarization ratio and uniaxial molecular order parameter were as high as 66 and 0.97, respectively. The aligned F8BT film was used as the active layer in a PLED, which resulted in polarized electroluminescence with a polarization ratio of 11. Ambipolar PFET in a top-gate configuration with aligned F8BT as the active semiconducting layer showed mobility enhancement when the chains were aligned parallel to the transport direction. Mobility anisotropies for hole and electron transport were 10-15 and 5-7, respectively, for current flow parallel and perpendicular to the alignment direction.
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103. Assembly of polyelectrolytes on CNTs by Van der Waals interactions and fabrication of LBL polyelectrolyte/CNT composites
We have successfully coated CNTs with either positively or negatively charged polyelectrolytes employing Van der Waals interactions between nanotubes and polyelectrolyte molecules, without any previous chemical modification of the nanotubes walls. Polymer adsorption of polyelectrolytes was performed in an ethanolic solution, followed by redispersion in water. The surface coating was verified with potential measurements and SEM. A sequential assembly of polyelectrolyte layers on top of the polyelectrolyte modified nanotubes has been achieved from a water solution following the layerby-layer technique. The assembly of polyelectrolytes from water on CNTs deposited on a surface has also been shown with OCM-D. Then, polyelectrolyte coated CNTs have been used as positive and negative blocks in the templating of layer-by-layer films.
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102. Synthesis and characterization of poly(3-sulfopropylmethacrylate) brushes for potential antibacterial applications
This article describes the aqueous atom transfer radical polymerization synthesis of poly(3-sulfopropylmethacrylate) brushes onto gold and Si/SiO2 surfaces in a controlled manner. The effect of Cu(I)/Cu(II) ratio was examined, and a quartz crystal microbalance was used to study the kinetics of the brush synthesis. The synthesized brushes displayed a thickness from a few nanometers to several hundred nanometers and were characterized using atomic force microscopy, ellipsometry, Fourier transform infrared spectroscopy (FTIR), contact angle measurements, and X-ray photoelectron spectroscopy (XPS). The as-synthesized sulfonate brushes had very good ion-exchange properties for the ions tested in this study, i.e., Na+, K+, Cu2+, and Ag+. FTIR and XPS show that the metal ions are coordinating to sulfonate moieties inside the brushes. The brushes were easily loaded with silver ions, and the effect of silver ion concentration on silver loading of the brush was examined. The silver-loaded brushes were shown to be antibacterial toward both gram negative and gram positive bacteria. The silver leaching was studied through leaching experiments into water, NaNO3, and NaCl (physiological medium). The results from these leaching experiments are compared and discussed in the article.
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101. Formation of hybrid 2D polymer-metal microobjects
This paper describes a fabrication strategy based on polymer brushes (20-150 nm thick) and soft lithographic techniques, for creating hydrophobic, cross-linked, laterally patterned polymer films. The hydrophobicity of the resulting micrometer-scale quasi-2D objects is shown to allow the polymer to act as an etch resist. By adjusting the etching time, we demonstrate that underetching of the gold from underneath the edges of the laterally patterned films can be used to create free-standing polymer-gold hybrid structures. These structures retain their structural integrity when lifted wholly or partially from the substrate and can hence be imaged in suspension. Characterization of the quasi-2D objects was carried out using atomic force microscopy (AFM), ellipsometry, optical microscopy, and Fourier transform infrared spectroscopy (FTIR). A continuous film, containing embedded polymer-gold objects, can be lifted, folded, and re-deposited onto a substrate without damaging the conductivity of the embedded metallic objects.
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100. Tunable wettability by clicking into polyelectrolyte brushes
Polymer brushes have recently emerged as an extremely versatile way to modify surface properties in a robust and controlled way. The introduction of responsive polymers and block copolymers in polymer-brush systems has also opened up new routes to ‘smart’ surfaces with switchable surface properties. here, the use of polyelectrolyte brushes as a supramolecular platform for the immobilization of a wide range of species, leading to a tunable wettability of substrates, is presented.
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99. Probing the responsive behavior of polyelectrolyte brushes using electrochemical impedance spectroscopy
Cyclic voltammetry and impedance spectroscopy were employed to probe the responsive properties of polyelectrolyte brushes. Poly[(dimethylamino)ethyl methacrylate] (PDMAEMA) brushes over 100 nm thick on gold substrates were synthesized via surface-initiated atom-transfer radical polymerization and quaternized with methane iodide to obtain cationic brushes (Q-PDMAEMA). Q-PDMAEMA brushes respond to electrolytes by exhibiting swollen and collapsed states. Swollen brushes allow good permeability of electroactive probes, while collapsed states block electron transport. Electrolytes have different impacts on the electrochemical properties of Q-PDMAEMA. Some salts (NaNO3) cause brush collapse due to charge screening, while others such as those with more hydrophobic anions (ClO4-, PF6-, and Tf2N-) induce brush collapse because of solubility changes. The collapsed brushes exhibit intrinsically different resistance as probed with impedance. Charged screened brushes retain good permeability to electroactive probes. Strongly coordinating hydrophobic anions lead to insoluble brushes, resulting in a high resistance. These results show that electrochemical impedance spectroscopy is a powerful technique to probe the properties and structure of polyelectrolyte brushes.
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98. Self-assembly meets nanofabrication: Recent developments in microcontact printing and dip-pen nanolithography
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97. A self-assembly approach to chemical micropatterning of poly(dimethylsiloxane)
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96. Synthesis of raspberry-like particles using polyelectrolyte multilayer-coated particles
The formation of raspberry particles from polyelectrolyte multilayer-modified particles is described. This synthesis represents a new use for polyelectrolyte multilayers as nanoscale-thick reaction vessels for the immobilization and reaction of inorganic reagents. The dissolution of Au nanoparticles inside PEM layers that are capped with a SiO2 layer leads to the hydrolysis and subsequent redeposition of SiO2 nanoparticles. The size of these particles is in the 200 nm range and their loading can be controlled by adding various amounts of Au nanoparticles to the PEMs.
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95. Surface-induced droplet fusion in microfluidic devices
Here we demonstrate a new method for droplet fusion based on a surface energy pattern on the walls of a microfluidic device, that does not require active elements nor accurate synchronization of the droplets.
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94. Polymer phase separation on lattice patterned surfaces
We demonstrate the self-organization of phase-separated polymer microstructures on two-dimensionally chemically patterned surfaces. Pattern replication is expected when both the blend composition ratio matches the surface patterning area ratio, and the pattern periodicity matches the natural phase separation length scale. By varying film thickness and blend composition, we show that ordered morphologies also result for contrary situations, resulting in the formation of a rich variety of hierarchically-ordered microstructures. This hierarchy suggests that non-equilibrium structures generated by incomplete phase separation of blend components are locked in by rapid solvent quenching during casting.
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93. Multicomponent polymer brushes
This article describes a general synthetic route to laterally distinctive multicomponent polymer brushes on gold. The procedure involves repeated surface patterning using microcontact printing (mu CP) of initiator-terminated thiols without backfilling with inert thiols and surface-initiated atomic transfer radical polymerization steps. In between brush growth, the remaining initiator moieties are deactivated to avoid reinitiation on existing brushes. Optical and fluorescence microscopy, atomic force microscopy, attenuated total reflectance Fourier transform infrared spectroscopy, and X-ray photoelectron spectroscopy have been used to characterize every step of this procedure. We found that brushes can be grown from initiator-modified surfaces that contain bare gold areas and that these areas remain available for further patterning using mu CP. To demonstrate the flexibility of this approach, surfaces containing four different polymer brushes in patterns ranging from 2 x 4 mu m lines to 20 x 20 mu m squares were fabricated. The range of chemical functionalities incorporated includes cationic and anionic polyelectrolytes, as well as thermally responsive polymers.
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92. The effect of [Cu-I]/[Cu-II] ratio on the kinetics and conformation of polyelectrolyte brushes by atom transfer radical polymerization
The atom transfer radical polymerization of [2-(methacryloyloxy)ethyl]trimethylammonium chloride (METAC) has been studied under different [Cu-I]/[Cu-II] ratios. The reaction kinetics is followed by ellipsometry and quartz crystal microbalance and it was found that the reaction speed influences the grafting density of the polymer brushes. High [Cu-I]/[Cu-II] ratios, i.e., fast polymerizations, lead to less dense polymer brushes.
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91. PMSE 222-Solid state self-assembly of polyferrocenylsilane diblock copolymers and applications in materials science
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90. PMSE 8-Hydrogen initiation of free radical polymerization under catalytic chain transfer conditions
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89. Stick and slide ferrofluidic droplets on superhydrophobic surfaces
The authors report the suspension and movement of ferrofluid droplets on superhydrophobic surfaces in the presence of strong external magnetic fields. The system allows the quantitative evaluation of friction between droplet and substrate surface. It represents an enabling example for the development of microferrofluidic devices and a breakthrough in the remote actuation of droplets on surfaces, which cannot be achieved using conventional microfluidics, or inkjet printing, electrowetting, or light-driven motion.
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88. Polyelectrolyte brushes as efficient ultrathin platforms for site-selective copper electroless deposition
Ion-exchange in surface-initiated polyelectrolyte brushes provides a versatile route to the formation of catalytically active surfaces for electroless deposition of Cu. The advantage of this procedure is the covalent anchoring of the catalyst support layer, eliminating delamination of the metal film, even when deposited onto PDMS substrates. Furthermore, by tuning the concentration of PdCl42- ions in the brushes, the rate of deposition and hence the thickness of the overall film can be controlled easily.
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87. Temperature-responsive glycopolymer brushes synthesized via RAFT polymerization using the Z-group approach
Stimuli-responsive glycopolymer brushes composed of N-acryloyl glucosamine (AGA) and N-isopropylacrylamide (NIPAAm) were prepared using RAFT polymerization. The RAFT agent was immobilized on the surface of a treated silicon waver via covalent attachment using the Z-group. PAGA and PNIPAAm brushes showed a linear increase in brush thickness with the consumption of monomer in solution. The polymers generated in solution confirm the living behavior with the molecular weight increasing linearly with monomer conversion while the molecular weight distribution remains narrow. Additionally, the ability of PAGA brushes to grow further in the presence of NIPAAm reveals the presence of an active RAFT end group indicative of a living system. PAGA and PNIPAAm homopolymer brushes up to 30 nm were grown using this technique. PAGA brushes were utilized for further chain extension to generate stimuli-responsive brushes with block structures of PAGA and PNIPAAm. the PAGA-block-PNIPAAm brushes were found to grow in size with the consumption of NIPAAm. Contact angle measurements confirm the suggested mechanism showing the the second monomer is incorporated between the first layer and the silicon surface as expected using the Z-group approach.
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86. Topography printing to locally control wettability
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85. Polyelectrolyte brushes as ink nanoreservoirs for microcontact printing of ionic species with poly(dimethyl siloxane) stamps
In this work a new variation of microcontact printing is described, which is used to transfer chemical patterns onto different substrates. The approach is based on the use of conventional elastomeric stamps modified with polyelectrolyte brushes. It is demonstrated that, by using stamps modified with brushes acting as preconcentrating/sorbent nanolayers, it is possible to control the uptake of aqueous inks containing ionic species. This controlled uptake can be easily used for site-selective delivery of the loaded species by means of soft lithography. The potential of this approach is demonstrated by creating patterned counterion domains in a flat polyelectrolyte brush and by promoting a site-selective metallization through galvanic displacement reactions with reactive species.
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84. Sub-10-nm high aspect ratio patterning of ZnO in a 500 mu m main field
We demonstrate the fabrication of sub-10-nm high aspect ratio electron beam patterning of ZnO at the center as well as corners of the 500 mu m square main deflection field using a negative tone zinc naphthenate resist. After electron beam exposure, the resist was developed in toluene, resulting in high-resolution patterns as small as 7 urn with an aspect ratio of similar to 10 in the center as well as at the corners of the main field. The line edge roughness (3 sigma) of these lines shows roughness with fluctuations of about 2.8 nm; thus making it the smallest value of the line edge roughness measured so far for any electron beam resist. Heating the structures at 500 degrees C resulted in reduction of feature sizes to similar to 5 nm and in the formation of crystalline ZnO. Such sub-10-nm high aspect ratio patterning within the main deflection field is an important step to enable full-wafer-level nanofabrication. (c) 2006 American Vacuum Society.
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83. Highly reversible and multi-stage cantilever actuation driven by polyelectrolyte brushes
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82. Transfer printing water-soluble inorganic salts
This paper reports the first example of the fabrication of KNO3, K2CO3, CuSO4, NaOH, and mixed-inorganic-salt (KNO3 and KOH) patterns using a transfer-printing (TP) technique. The transfer quality is found to be related to the concentration of the salt solutions. By varying the immersion time, it is possible to control the heights of the raised features of the transfer-printed salts from the nanoscale to the submicrometer scale. Utilizing these inorganic salts as water-soluble masks for microfabrication is demonstrated using patterned NaOH films. The use of water as a developer solvent demonstrates the potential utility of the patterning of inorganic salts as a low-cost. simple, and, more importantly, environmentally friendly route towards accurate patterning of different materials.
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81. Polymer brushes for soft nanotechnology
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80. Polyzwitterionic brushes: Switching based on self-association
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79. Buckling in quasi-2D polymers
Buckle-driven delamination and subsequent collapse of strained thin polymer films upon triggered release from the substrate is exploited to fabricate striking, well-defined ridging patterns (see figure). An analysis of these patterns is presented, including the effects of film thickness and the exterior shape of these quasi-2D polymer objects.
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78. Enhancement of charge-transport characteristics in polymeric films using polymer brushes
We show that charge-transporting polymer chains in the brush conformation can be synthesized from a variety of substrates of interest, displaying a high degree of stretching and showing up to a 3 orders of magnitude increase in current density normal to the substrate as compared with a spin-coated film. These nanostructured polymeric films may prove to be suitable for electronic devices based on molecular semiconductors as current fabrication techniques often provide little control over film structure.
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77. Thermal and UV shape shifting of surface topography
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76. A dendrimer-based Co-32 nanocluster: Synthesis and application in diameter-controlled growth of single-walled carbon nanotubes
We report the synthesis and characterization of a dendrimer-based Co-32 nanocluster molecule and its use as uniform-size catalytic seed for diameter-controlled growth of single-walled carbon nanotubes. (c) 2005 Elsevier Ltd. All rights reserved.
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75. UCST wetting transitions of polyzwitterionic brushes driven by self-association
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74. Mechanically induced generation of counterions inside surface-grafted charged macromolecular films: Towards enhanced mechanotransduction in artificial systems
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73. The electrolyte switchable solubility of multi-walled carbon nanotube/ionic liquid (MWCNT/IL) hybrids
In this communication we report the first preparation of ionic liquid-modified carbon nanotubes with reversibly switchable solubility between aqueous and organic solvents, induced by anion exchange.
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72. Surface grafted polymer brushes as ideal building blocks for ”smart” surfaces
Polymer brushes are assemblies of macromolecules chemically tethered at one end to a substrate. They provide an alternative to self-assembled monolayers because of the intrinsically large size of the building blocks and the ensuing entropic contribution to the film morphology. In this article, an overview of a number of representative polymer brush systems will be presented and their potential application for surfaces with controlled wettability, smart surfaces and nanoactuators will be explored in some detail.
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71. Catalyst patterning methods for surface-bound chemical vapor deposition of carbon nanotubes
We present three different catalyst preparation and patterning techniques for plasma-enhanced chemical vapor deposition of carbon nanostructures from acetylene and ammonia mixtures. The different merits and potential areas of application are highlighted for each technique as compared to the benchmark of e-beam-lithography patterning. Maskless, focused ion beam written Pt can nucleate aligned carbon nanofibers, thereby allowing a sub-100 nm lateral resolution on non-planar substrate geometries combined with an in-situ monitoring. Ion beam milling additionally enables the pre-shaping and marking of the substrate, which is shown for the growth of individual nanofibers on the apex of commercial scanning probe tips. Pulsed electrochemical deposition was used to form Ni and Fe catalyst islands of controlled size and density. This is also demonstrated on complex substrate geometries such as carbon cloth. Nanocontact printing was employed to deposit a highly purified Co colloid in regular patterns with feature sizes down to 100 nm onto silicon wafers for low cost patterning over large areas. We analyze the catalyst restructuring upon exposure to elevated temperatures for each technique and relate this to the nucleated nanofiber dimensions and array densities. The flexibility in catalyst and substrate material allows a transfer of our achievements to catalyst-assisted growth of nanostructures in general facilitating their hierarchical device integration and future application.
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70. Switching the properties of polyelectrolyte brushes via Hydrophobic collapse
We have studied the changes in physical and chemical properties of cationic poly(2(methacryloyloxy)ethyltrimethylammonium chloride) brushes after collapse driven by ion-pairing interactions in the presence Of ClO4- anions. Results derived from the quartz crystal microbalance technique, atomic force microscopy, Fourier transform infrared spectroscopy, and contact angle goniometry indicate that ion-paired collapsed polyelectrolyte brushes suffer a dramatic loss of water accompanied by conformational changes leading to markedly different mechanical properties. This scenario is completely different from polyelectrolyte brushes whose collapse is simply driven by pure Coulombic screening, for example, in the presence of Cl- anions. In addition, wetting measurements indicated that ion-pairing interactions can be used to switch surface characteristics from hydrophilic to hydrophobic in a reversible manner. The immediate implications of these experimental results are related to the promising use of polyelectrolyte brushes as biolubricants and the design of smart surfaces exhibiting ion-sensitive reversible changes in interfacial properties.
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69. Self-organization of nanocrystals in polymer brushes. Application in heterojunction photovoltaic diodes
We present a new approach to achieving order in molecular semiconductors via alignment of polymer chains using surf ace-initiated polymerization. Polyacrylate brushes grown from transparent conducting electrodes, with triarylamine side groups as hole-transporting components, show characteristics of high mobilities for hole transport. Solution processing a second component with favorable enthalpic interactions can form a composite with mesoscale order and be exploited for heterojunction diodes. We find substantial uptake of CdSe nanocrystals (with diameter in the range 2.5-2.8 nm), and such composites show photovoltaic quantum efficiencies of up to 50%.
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68. Submicron patterning of Co colloid catalyst for growth of vertically aligned carbon nanotubes
Applications of carbon nanotubes such as field emission or microelectrode sensor arrays require a patterning of vertically aligned carbon nanotubes over large areas. A highly purified and concentrated monodisperse cobalt colloid was produced for use as a catalyst for growth of carbon nanotubes. Nanocontact printing was employed to deposit the cobalt nanoparticles in regular patterns with feature sizes at the 100 nm scale onto silicon wafers at low cost over large areas. Vertically aligned carbon nanotubes were grown by direct current plasma enhanced chemical vapour deposition at temperatures ranging from 300 to 640 degrees C.
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67. Polymer brushes for responsive biocompatible surfaces
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66. Polyelectrolyte brushes: Switching between hard and soft matter
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65. Synthesis of oligo(ethylene glycol) methacrylate polymer brushes
Here we report a study into controlling the polymerization of mono-hydroxy and mono-methoxy terminated oligo(ethylene glycol) methacrylates (HOEGMA and MeOEGMA, respectively) from functionalised, planar surfaces via atom transfer radical polymerization (ATRP). The effects of initiator structure, initiator density, temperature, and monomer ratios have been investigated for these polymerizations. The polymer brushes grown in this way were found to convey protein resistance to the underlying inorganic substrates, prone to facile protein adsorption in their native state. (c) 2005 Elsevier Ltd. All rights reserved.
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64. Following polymer brush growth using the quartz crystal microbalance technique
The growth of surface-initiated poly([2-(methacryloyloxy)ethyl]trimethylammonium chloride) (pMETAC) brushes by ATRP was monitored by the quartz crystal microbalance technique with dissipation (QCM-D). The change in mass of the quartz crystals starting from the adsorption of a thiol initiator monolayer through to the growth of the polymer brushes was determined. The use of QCM-D allowed determination of the kinetics of polymerization from the surface. The technique can be applied to other polymers synthesised from surfaces and allows the study of varying conditions on the polymerization kinetics.
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63. Sub-10 nm high-aspect-ratio patterning of ZnO using an electron beam
An electron-beam-sensitive zinc naphthenate resist is used to pattern ZnO. Features as small as 7 nm and with an aspect ratio of similar to 9 can be patterned. Size reduction to similar to 5 nm is observed when these patterns are heat treated to give crystalline ZnO (see Figure). The functionality of ZnO is confirmed via photoluminescence studies.
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62. AFM study of cationically charged polymer brushes: switching between soft and hard matter
AFM studies on cationic polymer brushes in water (a good solvent) show that brushes in an extended conformation can be easily deformed or indented by an AFM tip. Experiments on polymer brushes in a more collapsed conformation, using methanol-water as a `poor solvent’ environment, show similar properties. Conversely, this `soft’ behaviour is dramatically different in the presence of electrolytes containing anions that are strongly coordinated to cationic groups of the polymer brush. Our initial studies in electrolyte solutions at different concentrations show that these brushes become so rigid that they cannot be indented or deformed by the AFM tip, even at high loads.
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61. Shape-memory nanoparticles from inherently non-spherical polymer colloids
Samples of polymeric materials generally have no intrinsic shape; rather their macroscopic form is determined by external forces such as surface tension and memory of shear ( for example, during extrusion, moulding or embossing). Hence, in the molten state, the thermodynamically most stable form for polymer ( nano) particles is spherical. Here, we present the first example of polymer nanoparticles that have an intrinsic non-spherical shape. We observe the formation of high-aspect-ratio ellipsoidal polymer nanoparticles, of controlled diameter, made from main-chain liquid crystalline polymers using a mini-emulsion technique. The ellipsoidal shape is shown to be an equilibrium ( reversible) characteristic and a direct result of the material shape memory when a liquid crystal nanoparticle is in its monodomain form.
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60. Artificial skins – Hierarchical wrinkling
Wrinkling is a ubiquitous form of mechanical instability, occurring in such widely different systems as skin and lava flows. Hierarchical wrinkling leading to topographical features, with length scales spanning five orders of magnitude, has now been observed and harnessed in an artificial skin.
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59. Nanostructured polymers in polymeric optoelectronic devices.
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58. Polymer brushes: From synthesis to ‘smart surfaces’.
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57. Homeotropic alignment on surface-initiated liquid crystalline polymer brushes
With the development of new types of liquid crystal displays and the use of liquid crystals as organic semiconductors, homeotropic alignment layers have become more and more important. Liquid crystalline polymer (LCP) brushes have been Suggested as an alignment layer and are expected to have more interaction with the liquid crystal phase than other homeotropic alignment layers. Here we report the synthesis of side chain liquid crystalline polymer brushes by surface-initiated Atom Transfer Radical Polymerisation (ATRP) of an acrylate functionalized mesogen on glass and silicon substrates. Brush thicknesses up to 20 run were achieved. Substrates coated with a brush layer of this polymer were used for alignment of the nematic liquid crystal 5-cyanobisphenyl (5CB). Nearly defectless homeotropic alignment over several square centimetres is demonstrated. Microcontact printing has been used to obtain a patterned alignment layer.
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56. Locking and unlocking of polyelectrolyte brushes: toward the fabrication of chemically controlled nanoactuators
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55. Organisation of recombinant lipid-tagged cytochrome on surface
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54. Effects of nanoconfinement on the morphology and reactivity of organic materials
When organic materials are placed in environments which physically confine the materials at the nanometre scale, interfacial effects and confinement-induced loss of entropy can significantly alter materials’ properties such as the glass transition temperature as well as the nanoscale morphology as compared to a ‘free’ system. In block copolymers, nanoconfinement leads to a range of unusual self-organized nanoscale morphologies. In this article, attempts to induce nanoconfinement effects in new polymer systems as well as at interfaces will be highlighted and some possible future implications for organic synthesis and biology will be discussed.
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53. Three-stage switching of surface wetting using phosphate-bearing polymer brushes
Surface initiated polymer brushes containing phosphate side groups have been used to reversibly switch wettability and hydration between three different states.
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52. Bioadhesion at micro-patterned stimuli-responsive polymer brushes
The synthesis of poly(N-isopropylacrylamide) brushes within micropatterned domains at surfaces and the performance of these functionalised surfaces in short-term bioadhesion assays under varying conditions are described. The polymer brushes show temperature dependent behaviour at surfaces as demonstrated by changes in contact angle, surface energy components and aqueous phase AFM. The responses in the polymer brush domains result in spatially defined, and temperature mediated, attachment of a model protein, BSA, and the common oral bacteria Streptococcus mutans.
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51. Self-organized photonic structures in polymer light-emitting diodes
The creation of photonic structures in thin polymer films is reported. This involves replicating a micrometer-sized pattern onto a (poly(ethylenedioxythiophene) doped with polystyrene sulfonic acid) PEDOT:PSS surface (see Figure) with conjugated polymers without compromising the efficiencies of the devices. The technique can be extended to flexible substrates, and in principle to conjugated polymer blends.
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50. Polymer field effect transistors fabricated by dewetting
Electronic industry in polymer transistor circuits requires patterning techniques that are capable of defining critical features down to submicrometer- or even nanometer-scale resolution. Employing conducting polymer as electrodes instead of inorganic noble metals, is highly industry needed towards low-cost fabrication of plastic electronic chips. We developed a dewetting process for fabricating short-channel polymer field effect transistors with conducting polymer employed as electrodes. Source and drain contacts were patterned by splitting conducting polymer water solution by a hydrophobic self-assembled monolayer, elevated by a hydrophilic insulating mesa, with the channel length actually determined by the length of the mesa. Polymer transistors with channel length down to 500 nm have been fabricated. (C) 2004 Elsevier B.V. All rights reserved.
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49. Highly-efficient broadband waveguide outcoupling in light-emitting diodes with self-organized polymer blends
Self-organized, two-dimensional micron-scale photonic structures have been fabricated within the emissive layer of polymer blend light-emitting diodes (LEDs). The relief-and-phase grating is achieved by phase separation of two semiconducting polymers directed by a surface chemical pattern. Short-wavelength oscillations are found pinned (and are thus phase-locked) to the domain boundaries. These high-frequency harmonics mimic short-period gratings in providing efficient waveguide outcoupling but without spectral dispersion. This provides a general way to harness the waveguide modes trapped in polymer LEDs, doubling their external quantum and power efficiencies while maintaining spectral integrity with viewing angle. (C) 2004 American Institute of Physics.
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48. Ordered block-copolymer assembly using nanoimprint lithography
Nanoimprint lithography and self-assembly of poly(styrene)-block-poly(methyl methacrylate) (PS-b-PMMA) diblock copolymer are combined to induce order in the phase-separated domains. Tailored periodic arrays of poly(methyl methacrylate) (PMMA) cylinders normal or parallel to neutralized silicon surfaces can be formed inside the gap of imprint molds. This method opens up a new route to the controlled phase separation of block copolymers with precise placement of the phase-separated domains.
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47. Surface-synthesis of oligopeptides: Reactions in nanoconfinement.
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46. Quasi-2D polymer objects from patterned, crosslinked polymer brushes
Polymer sheets (see Figure) with nanometer-scale thicknesses, micrometer-scale lateral dimensions, and interesting materials properties that could be exploited in applications such as polymeric nanoactuators, biomimetic systems, and drug delivery vehicles have been fabricated via a new approach. The method employs crosslinked polymer brushes supported on micropatterned surfaces and subsequent electrolytic lifting.
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45. Synthesis of patterned polymer brushes from flexible polymeric films
Surface-initiated polymerizations were carried out from polymeric surfaces of commercially important polyester films, poly(ethylene terephthalate) (PET) and poly(ethylene naphthalate) (PEN). These plastic films were modified prior to polymerization by plasma oxidation, exposing surface hydroxyl groups, in order to immobilise patterned self assembled monolayers (SAMs) of trichlorosilane initiator, through the soft lithographic method of microcontact printing (muCP). Subsequently, polymerizations were initiated from the surface via controlled atom transfer radical polymerization (ATRP), under aqueous conditions, to create patterned brushes of the thermo-responsive polymer poly(N-isopropyl acrylamide) (PNIPAM). By creating patterned, rather than homogeneous brushes characterization was made possible by atomic force microscopy (AFM). (C) 2004 Elsevier Ltd. All rights reserved.
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44. Self-assembled monolayers (SAMS) and synthesis of planar micro- and nanostructures
A polymer is a macromolcule made up of multiple equivalents of one or more monomers linked together by covalent bonds. Self-assembled monolayers (SAMs) are highly ordered molecular assemblies formed by chemisorption of functionalized molecules on the surface. Various system of SAMs are reviewed in this article. The synthesis and structures of SAMs on gold and silver are briefly discussed along with chemical reactions that occur on SAMs after their assembly. The patterning of SAMs on gold and its applications are discussed in the plane of the monolayer.
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43. Carbon dioxide as a solvent for synthesis and processing
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42. Dewetting of conducting polymer inkjet droplets on patterned surfaces
The manufacture of high-performance electronic devices with micrometre or even submicrometre dimensions by solution processing and direct printing, requires the ability to control accurately the flow and spread of functional liquid inks on surfaces. This can be achieved with the help of surface-energy patterns causing inks to be repelled and dewetted from pre-defined regions of the substrate. To exploit this principle for the fabrication of submicrometre device structures, a detailed understanding of the factors causing ink droplets to dewet on patterned surfaces is required. Here, we use hydrophobic surface-energy barriers of different geometries to study the influence of solution viscosity, ink volume, and contact angle on the process of dewetting of inkjet-printed droplets of a water-based conducting polymer. We demonstrate polymer field-effect transistor devices with channel length of 500 nm fabricated by surface-energy-assisted inkjet printing.
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41. Controlled growth and subsequent chemical modification of poly(glycidyl methacrylate) brushes on silicon wafers
The controlled growth of poly(glycidyl methacrylate) (PGMA) and poly(GMA-co-MMA) brushes by atom transfer radical polymerization (ATRP) in methanol/water has been demonstrated on silicon wafer surfaces. A polymerization system using copper(I) bromide, copper(II) chloride and 2,2′-dipyridyl was found to produce a nearly linear rate of growth for times of up to 4 h, giving brushes of up to 120 nm thickness. Reinitiation of these brushes to grow further PGMA demonstrated the living nature of the polymerization system. A trichlorosilane-functional initiator was used, and it was found that brushes grown from initiator layers deposited in the presence of a small amount of triethylamine were less rough and showed more linear growth with time. The reaction of the pendant epoxide group of these brushes with octylamine from solution was demonstrated.
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40. Polymer brushes via surface-initiated polymerizations
Polymer brushes produced by controlled surface-initiated polymerization provide a route to surfaces coated with well-defined thin polymer films that are covalently bound to the substrate. All of the major controlled polymerization techniques have been applied to the synthesis of polymer brushes and examples of each are presented here. Many examples of brush synthesis in the literature have used the living atom transfer radical polymerization (ATRP) system, and in this tutorial review a particular focus is given to examples of this technique.
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39. Forced peptide synthesis in nanoscale confinement under elastomeric stamps
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38. A nanoscale dendrimer-based Fe-24 cluster: synthesis and molecular self-assembly
We report the synthesis, characterisation and self-assembly of a nanoscopic Fe-24 cluster using an organic dendrimer as a molecular substrate for building up well-defined molecular nanostructures.
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37. Nanostructured polymers
Polymers are ideal building blocks to fabricate organic nanostructures. To bridge the gap between natural and ‘fabricated’ devices more control over polymers at the nanometer level is required. This review will highlight a number of recent advances ill polymer synthesis, self-organization of block copolymers. and soft-lithographic techniques, that all provide new routes towards nanostructured polymers.
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36. Hyperbranched polyglycidol on Si/SiO2 surfaces via surface-initiated polymerization
We demonstrate a new procedure to synthesize covalently linked hyperbranched polyglycidol brushes on Si/SiO2 surfaces via anionic ring-opening multibranching polymerization of glycidol at 110 degreesC. Optimization of the polymerization experiments by exploiting reinitiation cycles produced polyglycidol brushes with ellipsometric thickness values of up to 70 nm. C-13 NMR spectroscopy analysis of cleaved polymer allowed the elucidation of the structure and degree of branching of the polymer.
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35. Fluorinated silane self-assembled monolayers as resists for patterning indium tin oxide
We report the formation of self-assembled monolayers (SAMs) on indium tin oxide (ITO) substrates with perfluoroorganosilanes in liquid and supercritical carbon dioxide and a method of patterning the monolayer that does not use any organic solvents. The monolayers formed were used as an etch resistant during the formation of patterns as small as 300 nm. The monolayers were characterized using wettability experiments, surface FT-IR, cyclic voltammetry, and AFM. The effects of temperature and adsorption time on the formation of SAMs were explored. Advancing contact angles as high as 105degrees and fractional surface coverages up to 0.96 were achieved by exposing the ITO surfaces to silanes in ScCO2 for 15 h. Surface FT-IR results show peaks at 1212 and 1152 cm(-1), typical for disordered monolayers. Yet, these SAMs are resistant to wet etching for over 10 h, indicating that dense carbon dioxide is a superior solvent for SAM formation of perfluorosilanes on ITO.
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34. Nanocontact printing: A route to sub-50-nm-scale chemical and biological patterning
This paper describes the first example of the patterning of surfaces via nanocontact printing with chemically distinct features in the <50 nm scale over 3 x 3 mm(2). These ultrasmall features are achieved via a combination of sharp and hard poly(dimethylsiloxane) stamps, keeping the contact area in the 50 nm domain, and high molecular weight inks to avoid diffusion. The patterns consist of dendrimers or proteins and can be used as a scaffold for further modification with gold nanoparticles.
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33. Hyperbranched polyglycidol brushes.
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32. Focused ion beam fabrication of silicon print masters
We investigated a focused ion beam nanofabrication technique as a high-resolution patterning method suitable for nanocontact imprinting. Different ion beam currents, milling times, and dwell times are exploited to optimize focused ion beam milling conditions. Single-pixel lines are milled on a silicon master and replicated on polydimethylsiloxane through replica moulding. The profile of the grooves (the depth-to-width aspect ratio) was found to be depth dependent regardless of the beam current and dwell time. The depth of the line cuts was strongly dependent upon beam current and dwell time at a given dose. This technique holds great promise for mass production of nanostructures due to its simplicity and high reproducibility.
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31. Reactions on monolayers: Organic synthesis in two dimensions
Self-assembled monolayers (SAMs) provide ideal molecularly defined platforms to study reactions in two dimensions. The surface chemistry of SAMs can easily be controlled by the head group of the surfactant molecules, which self-assemble into perfectly ordered, crystalline monolayers. A wide range of organic transformations can then be carried out to change the surface chemistry. These reactions have important applications in biological microarray fabrication, such as DNA and peptide chips. In this Microreview we will give an overview of all the classes of reactions that have been performed on SAMs. We will discuss the fundamental difficulties related to synthesis on monolayers, focussing on issues like steric hindrance, characterization, determination of yields and possibilities for multistep syntheses.
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30. Patterned deposition from compressed carbon dioxide
Compressed CO2 is employed as the solvent for the deposition of polymers onto patterned surfaces created by a lithographic technique. This deposition technique should have wide applicability in the deposition of organic and polymeric materials for optoelectronic devices. The advantage of controlled deposition confers a further benefit in the control of the patterned surface. In a specific example a perfluorinated polymer was dissolved in liquid carbon dioxide. The polymer solution was deposited by use of a nozzle onto a pre-patterned surface. The resulting polymer film showed a clear image of the original pattern as measured by optical microscopy.
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29. Variable adhesion of micropatterned thermoresponsive polymer brushes: AFM investigations of poly (N-isopropylacrylamide) brushes prepared by surface-initiated polymerizations
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28. Creating nanoscale patterns of dendrimers on silicon surfaces with dip-pen nanolithography
Dip-pen nanolithography (DPN) is a nanowriting procedure that employs an AFM tip as a nanopen to deposit organic molecules onto a substrate surface. This paper describes the application of DPN to write with dendrimer inks. We have generated patterns with 100 nm features (similar to20 dendrimer molecules) on a Si/SiOx surface and investigated how the resolution is affected by surface chemistry and molecular weight of the dendrimer ink.
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27. High-resolution contact printing with dendrimers
We have used amine-terminated polyamidoamine (PAMAM) dendrimer generation 4 as ink for contact printing. Periodic dendrimer lines with widths of 140 nm and interline widths of 70 nm were printed directly on a silicon substrate. Without obvious molecular diffusion during printing, the printed pattern is only determined by the conformal contact area, which is in turn determined by the mechanical properties of elastomeric stamp.
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26. Vertically segregated polymer-blend photovoltaic thin-film structures through surface-mediated solution processing
Surface treatment and solvent evaporation control are used to promote vertical segregation in polyfluorene-blend thin films. This surface-mediated control of the compositional structure in the direction normal to the plane of the film has important implications for optimizing charge transport in solution-processed conjugated polymer-blend optoelectronics. Here, the surface energy of the hole-collector electrode of photovoltaic devices is modified by deposition of self-assembled monolayers to favor segregation of the hole-accepting component of the blend to the substrate. Devices fabricated with intentionally vertically segregated blends showed external quantum efficiencies of up to 14%, which is ten times higher than that of devices fabricated without surface modification. (C) 2002 American Institute of Physics.
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25. Surface-initiated polymerizations in aqueous media: Effect of initiator density
We report the first systematic study of initiator density on surface-initiated polymerizations. We used mixed monolayers comprising of undecanethiol and omega-mercaptoundecyl bromoisobutyrate (1) to initiate the controlled radical polymerization of methyl methacrylate (MMA) and glycidyl methacrylate (GMA) from gold. We found that SAMs composed of 10% and 50% of initiator 1 grow PMMA brushes to approximately 1/10 and 1/2 the thickness of polymer brushes grafted from SAMs comprising 100% 1. We did not find a clear maximum initiator density, beyond which no further increase in polymer brush thickness is observed. We show that the initiator density at the start of the polymerization determines the ultimate footprint and hence density of polymer molecules in polymer brushes.
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24. Polymers in nanotechnology
The natural length scales of polymer chains and their morphologies in the bulk, which lie in the nanometer domain, make polymers ideal building blocks for nanotechnology. In this review we will discuss a number of recent developments in the use of polymers for the fabrication of nanostructures via lithographic and self-assembling strategies. (C) 2002 Elsevier Science Ltd. All rights reserved.
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23. Microembossing of elastomeric triblock copolymers
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22. Controlled growth of triblock polyelectrolyte brushes
We have achieved a significant breakthrough in the synthesis of polyelectrolyte brushes of controlled thickness and density, which has been demonstrated by the synthesis of triblock copolymer brushes composed of cationic, neutral, and anionic segments.
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21. Controlled surface-initiated polymerizations in aqueous media
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20. Patterning electro-osmotic flow with patterned surface charge (vol 84, pg 3314, 2000)
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19. Patterning electro-osmotic flow with patterned surface charge
This Letter reports the measurement of electro-osmotic flows (EOF) in microchannels with surface charge patterned on the 200 mu m scale. We have investigated two classes of patterns: (1) Those in which the surface charge varies along a direction perpendicular to the electric field used to drive the EOF; this type of pattern generates multidirectional flow along the direction of the field. (2) Those in which the surface charge pattern varies parallel to the field; this pattern generates recirculating cellular flew, and thus causes motion both parallel and perpendicular to the external field. Measurements of both of these flours agree well with theory in the Limit of thin double layers and low surface potential.
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18. Ordering of spontaneously formed buckles on planar surfaces
This paper describes the spontaneous formation of patterns of aligned buckles in a thin film of gold deposited on the surface of an elastomer [poly(dimethylsiloxane), PDMS]. The surface of the elastomer is patterned photochemically into areas differing in stiffness and coefficient of thermal expansion. The gold is deposited while the surface of the patterned elastomer is warm (T similar to 100 degrees C). On cooling, shrinkage in the elastomer places the gold film under compressive stress. The buckles relieve this compressive stress. The distribution of stresses and buckle patterns is described during the pre- and postbuckling regimes using solutions from calculations describing a model comprising a thin stiff plate resting on a thick elastic foundation.
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17. Synthesis of geometrically well defined, molecularly thin polymer films
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16. The controlled formation of ordered, sinusoidal structures by plasma oxidation of an elastomeric polymer
This letter describes a technique for generating waves on polydimethylsiloxane (PDMS) patterned in bas-relief. The PDMS is heated, and its surface oxidized in an oxygen plasma; this oxidation forms a thin, stiff silicate layer on the surface. When the PDMS cools, it contracts and places the silicate layer under compressive stress. This stress is relieved by buckling to form patterns of waves with wavelengths from 0.5 to 10 mu m. The waves are locally ordered near a step or edge in the PDMS. The wavelength, amplitude, and pattern of the waves can be controlled by controlling the temperature of the PDMS and the duration of the oxidation. The mechanism for the formation and order of the waves is described. (C) 1999 American Institute of Physics. [S0003-6951(99)01943-9].
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15. Patterned polymer multilayers as etch resists
This paper describes the synthesis and characterization of patterned polymer multilayers on self-assembled monolayers (SAMs) and the use of these structures as etch resists for gold. The procedure used to build polymer multilayers consisted of five steps: (i) A polar thiol-HS(CH2)(15)COOH-was patterned on gold or silver films by microcontact printing (mu CP) with a poly(dimethylsiloxane) (PDMS) stamp. (ii) The patterned surface was placed in a solution containing CH3(CH2)(15)SH to form a nonpolar, methyl-terminated SAM on the remaining bare metal surface. (iii) The regions of the SAM patterned with CO2H groups were activated for further chemical reaction by conversion into interchain anhydride groups. (iv) The activated substrate was allowed to react with poly(ethylene imine) (PEI, branched chain, M-w 750 000). (v) A second polymer layer was attached to the PEI layer by allowing the amine-terminated surface to react with poly(octadecene-alt-maleic anhydride) (POMA, M-w 30 000) or poly(styrene-alt-maleic anhydride) (PSMA, M-w 350 000). This procedure (alternating reaction with PEI and POMA/PSMA) was repeated up to five times to increase the number of layers and the thickness of the patterned structure. The polymer multilayers were characterized using atomic force microscopy (AFM), ellipsometry, and polarized infrared external reflectance spectroscopy (PIERS). The stability of the films was demonstrated by using the patterned polymer multilayers as etch resists and by measuring their breakdown voltages.
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14. Patterning thin films of poly(ethylene imine) on a reactive SAM using microcontact printing
This paper describes the patterning of poly(ethylene imine) (PEI) on a surface into structures having submicron edge resolution. This procedure consists of three steps: (1) formation of a reactive self-assembled monolayer (SAM) terminating in interchain carboxylic anhydride groups on gold and silver; (2) patterning of this SAM by microcontact printing (mu CP) using a poly(dimethylsiloxane) (PDMS) stamp inked with PEI (this polymer contains primary and secondary amines that are reactive toward the anhydride groups); (3) hydrolysis of the unreacted anhydride groups with base and removal of noncovalently bound PEI. The patterned thin films of PEI are attached covalently to the SAM by amide bonds. The pendant, unreacted primary and secondary amines of the attached PEL can be used as reactive nucleophilic groups in further steps of chemical modification. This type of postmodification has been illustrated by allowing the amine groups of the covalently attached PEI to react with perfluorooctanoyl chloride, palmitoyl chloride, palmitic anhydride, and poly(styrene-alt-maleic anhydride). The PEI films and their derivatives were characterized using atomic force microscopy (AFM), scanning electron microscopy (SEM), polarized infrared external reflectance spectroscopy (PIERS), contact angles of water, and X-ray photoelectron spectroscopy (XPS).
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13. Surface-confined metallodendrimers: Isolated nanosize molecules
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12. Three-dimensional mesoscale self-assembly
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11. Molecularly thin films of metallodendrimers
A non-covalent synthesis route to assemble metallodendrimers was extended to the fifth generation. Generation four (G(4)) was characterized by electrospray mass spectrometry. Thin films of generation five (G(5)) metallodendrimers were studied by tapping mode scanning force microscopy. Both on graphite and on mica spherical particles with a diameter of ca 15 nm were observed. (C) 1998 John Wiley gr Sons, Ltd.
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10. Convergent and divergent noncovalent synthesis of metallodendrimers
A new building block is constructed with one pyridine and two kinetically inert complexed Pd(II) ions, for the controlled assembly of metallodendrimers following either a convergent or a divergent route. The double pincer ligand 8 was cyclopalladated with Pd[CH3CN](4)(BF4)(2) and subsequently converted into the neutral bis-palladium chloride complex 3. The pyridine moiety of 3, that is covalently attached to the spacer bridging the two pincer complexes, coordinates to activated palladium centers. Via a combination of pyridine(3) and cyano-based (2) building blocks, dendrons up to generation three were assembled and characterized with H-1 NMR and FT-W spectroscopy and MALDI-TOF mass spectrometry. These dendrons can coordinate through a cyano Ligand to an activated nucleus 1 forming convergently assembled metallodendrimers. Alternatively, building blacks 3 were used in the divergent assembly of more stable metallodendrimers, because of the stronger coordination of pyridine compared to cyano ligands. The formation of metallodendrimers is evidenced by IR and H-1 NMR spectroscopy and electro spray and MALDI-TOF mass spectrometry.
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9. Nanosize metallodendrimers
Building blocks have been designed for the non-covalent formation of nanosize assemblies. As non-covalent interactions coordination chemistry and hydrogen bonding have been used. The self-assembly process leads to spherical assemblies with diameters in the range of 100 to 400 nm, with standard deviations in the order of 10-15 %. The controlled assembly approach allows a precise controll of size and assemblies with molecular weights up to 10,000 Dalton have been realized. Finally, we have developed a strategy in which hydrogen bonding and coordination chemistry can be applied orthogonally.
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8. Non-covalent synthesis of multiporphyrin systems
Non-covalent synthesis was used in the controlled assembly of metallodendrimers containing up to 12 porphyrins on the surface. A porphyrin containing four Pd-Cl complexes was synthesized to assemble dendrimers with a porphyrin in the nucleus. H-1 NMR, ES-MS and MALDI-TOF mass spectrometry were used to characterize the nanometer-size assemblies.
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7. New building blocks for the noncovalent assembly of homo- and hetero-multinuclear metallodendrimers
Ligands 5 and 8, derived from the coupling of [3,5-bis[(diphenylphosphinyl)methyl]phenyl] oxy groups to bi- and trifunctional spacers, respectively, were cyclometalated with Pd[CH3CN](4)(BF4)(2), cis-[PtCl2(PPh3)(2)], or NiCl2 . 6H(2)O. The cationic Pd and Pt complexes were converted into overall neutral Pd-Cl and Pt-Cl complexes. The resulting pincer complexes can be used as building blocks for the controlled assembly of both homo- and hetero-multinuclear metallodendrimers. The P-31 NMR spectra of the pincer complexes exhibit different shifts for chloride, H2O, or nitrile occupying the fourth coordination site, making the P-31 chemical shift an excellent diagnostic tool for the assembly process.
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6. Self-assembly of hyperbranched spheres
A new type of building block with two coordinatively unsaturated palladium centres has been described that self-assembles in nitromethane solution and disassembles when acetonitrile is added. The resulting hyperbranched, organopalladium spheres have a remarkably narrow size distribution as was evidenced by light-scattering, AFM and TEM measurements. Variation of the structure of the building blocks showed the possibility to vary the size of the self-assembled spheres between 100 and 400 nm.
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5. Noncovalent synthesis of nanostructures: Combining coordination chemistry and hydrogen bonding
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4. Self-assembly of hyperbranched spheres; Correlation between monomeric synthon and sphere size
Light-scattering experiments show that the size of self-assembled hyperbranched spheres can be varied from 100 to 400 nm, by variation of the building block structure and/or the counter anions.
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3. Controlled assembly of nanosized metallodendrimers
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2. LARGE SELF-ASSEMBLED ORGANOPALLADIUM SPHERES
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1. SYNTHESIS OF NEW MACROCYCLIC LIGANDS FOR HETERO-MULTINUCLEAR TRANSITION-METAL COMPLEXES
The [3 + 3] macrocyclic compounds 2a-2c were easily prepared by cyclization of the dialdehyde 8 with the appropiate diamines in refluxing CH3CN/MeOH (1/1), in the presence of Ba2+ as a template ion. These macrocycles contain three salen units and are therefore, in combination with the crown ether interior, potentially tetranucleating. The length of the diamine bridge and the temperature at which the reaction is carried out, appear to influence the size of the macrocycle formed. In this way, the [4 + 4] macrocycle 2d could be formed selectively over the [3 + 3] macrocycles by carrying out the reaction at room temperature.