Publications by year
In Press
Dettmer SL, Pagliara S, Misiunas K, Keyser UF (In Press). Anisotropic diffusion of spherical particles in closely confining. microchannels.
Physical Review E,
98Abstract:
Anisotropic diffusion of spherical particles in closely confining. microchannels
We present here the measurement of the diffusivity of spherical particles
closely confined by narrow microchannels. Our experiments yield a
two-dimensional map of the position-dependent diffusion coefficients parallel
and perpendicular to the channel axis with a resolution down to 129 nm. The
diffusivity was measured simultaneously in the channel interior, the bulk
reservoirs, as well as the channel entrance region. In the channel interior we
found strongly anisotropic diffusion. While the perpendicular diffusion
coefficient close to the confining walls decreased down to approximately 25% of
the value on the channel axis, the parallel diffusion coefficient remained
constant throughout the entire channel width. In addition to the experiment, we
performed finite element simulations for the diffusivity in the channel
interior and found good agreement with the measurements. Our results reveal the
distinctive influence of strong confinement on Brownian motion, which is of
significance to microfluidics as well as quantitative models of facilitated
membrane transport.
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Misiunas K, Pagliara S, Lauga E, Lister JR, Keyser UF (In Press). Non-decaying hydrodynamic interactions along narrow channels.
Phys. Rev. Lett.,
115Abstract:
Non-decaying hydrodynamic interactions along narrow channels
Particle-particle interactions are of paramount importance in every
multi-body system as they determine the collective behaviour and coupling
strength. Many well-known interactions like electro-static, van der Waals or
screened Coulomb, decay exponentially or with negative powers of the particle
spacing r. Similarly, hydrodynamic interactions between particles undergoing
Brownian motion decay as 1/r in bulk, and are assumed to decay in small
channels. Such interactions are ubiquitous in biological and technological
systems. Here we confine two particles undergoing Brownian motion in narrow,
microfluidic channels and study their coupling through hydrodynamic
interactions. Our experiments show that the hydrodynamic particle-particle
interactions are distance-independent in these channels. This finding is of
fundamental importance for the interpretation of experiments where dense
mixtures of particles or molecules diffuse through finite length, water-filled
channels or pore networks.
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Tan Y, Dagdug L, Gladrow J, Keyser UF, Pagliara S (In Press). Particle transport across a channel via an oscillating potential.
Phys. Rev. E,
96Abstract:
Particle transport across a channel via an oscillating potential
Membrane protein transporters alternate their substrate-binding sites between
the extracellular and cytosolic side of the membrane according to the
alternating access mechanism. Inspired by this intriguing mechanism devised by
nature, we study particle transport through a channel coupled with an energy
well that oscillates its position between the two entrances of the channel. We
optimize particle transport across the channel by adjusting the oscillation
frequency. At the optimal oscillation frequency, the translocation rate through
the channel is a hundred times higher with respect to free diffusion across the
channel. Our findings reveal the effect of time dependent potentials on
particle transport across a channel and will be relevant for membrane transport
and microfluidics application.
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2020
Stie MB, Corezzi M, Juncos Bombin AD, Ajalloueian F, Attrill E, Pagliara S, Jacobsen J, Chronakis IS, Nielsen HM, Foderà V, et al (2020). Waterborne Electrospinning of α-Lactalbumin Generates Tunable and Biocompatible Nanofibers for Drug Delivery.
ACS Applied Nano MaterialsAbstract:
Waterborne Electrospinning of α-Lactalbumin Generates Tunable and Biocompatible Nanofibers for Drug Delivery
© 2020 American Chemical Society. Protein-based drug carriers are an interesting alternative to traditional polymeric drug delivery systems due to their intrinsic biocompatibility and biodegradability. Electrospinning of neat proteins holds advantages over electrospinning of protein mixtures, e.g. whey isolates, such as better control of the physicochemical and biological function of the resulting nanofiber-based system. In this study, we explore electrospinning of the isolated milk protein α-lactalbumin (ALA), which is a whey protein with important nutritional and pharmacological properties. Via waterborne electrospinning of ALA with a minimum amount of poly(ethylene oxide) (PEO) as a cospininng polymer, nanofibers of high protein content were successfully produced (up to 84% (w/w)). We demonstrate the ability to produce ALA-based nanofibers with a high degree of tunability in terms of size, stability in water, and mechanical properties. The nanofibers displayed excellent biocompatibility in vitro as the viability of cultured TR146 human buccal epithelium and NIH 3T3 murine fibroblast cells was not influenced by exposure to ALA-based nanofibers. ALA-based nanofibers were loaded with up to 6% (w/w) ampicilin, and the nanofibers were capable of maintaining the activity of the antibiotic after electrospinning and cross-linking. Using such a property of the material, we demonstrate that ampicillin-loaded nanofibers successfully inhibit the growth of Gram-negative bacteria in vitro. Importantly, after treatment with ampicillin-loaded nanofibers, no bacterial regrowth was observed, which indicates that this treatment may clear eventual persisters to ampicillin. Finally, the structural properties of the native functional protein were maintained after release of ALA from the nanofibers. This promotes our platform, not only as a sustainable protein-based drug delivery system, but also as an innovative solid form of ALA for food and pharmaceutical applications.
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2019
Martin EL, Bryan MT, Pagliara S, Ogrin FY (2019). Advanced Processing of Micropatterned Elasto-Magnetic Membranes.
IEEE Transactions on Magnetics Full text.
Blaskovich MA, Phetsang W, Stone MRL, Lapinska U, Pagliara S, Bhalla R, Cooper MA (2019). Antibiotic-derived molecular probes for bacterial imaging.
Abstract:
Antibiotic-derived molecular probes for bacterial imaging
Abstract.
Łapińska U, Glover G, Capilla-Lasheras P, Young AJ, Pagliara S (2019). Bacterial ageing in the absence of external stressors.
Philosophical Transactions of the Royal Society B: Biological Sciences,
374(1786), 20180442-20180442.
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Kepiro IE, Marzuoli I, Hammond K, Ba X, Lewis H, Shaw M, Gunnoo SB, De Santis E, Łapińska U, Pagliara S, et al (2019). Engineering Chirally Blind Protein Pseudocapsids into Antibacterial Persisters.
ACS NanoAbstract:
Engineering Chirally Blind Protein Pseudocapsids into Antibacterial Persisters.
Antimicrobial resistance stimulates the search for antimicrobial forms that may be less subject to acquired resistance. Here we report a conceptual design of protein pseudocapsids exhibiting a broad spectrum of antimicrobial activities. Unlike conventional antibiotics, these agents are effective against phenotypic bacterial variants, while clearing "superbugs" in vivo without toxicity. The design adopts an icosahedral architecture that is polymorphic in size, but not in shape, and that is available in both l and d epimeric forms. Using a combination of nanoscale and single-cell imaging we demonstrate that such pseudocapsids inflict rapid and irreparable damage to bacterial cells. In phospholipid membranes they rapidly convert into nanopores, which remain confined to the binding positions of individual pseudocapsids. This mechanism ensures precisely delivered influxes of high antimicrobial doses, rendering the design a versatile platform for engineering structurally diverse and functionally persistent antimicrobial agents.
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Smith A, Metz J, Pagliara S (2019). MMHelper: an automated framework for the analysis of microscopy images acquired with the mother machine.
Sci Rep,
9(1).
Abstract:
MMHelper: an automated framework for the analysis of microscopy images acquired with the mother machine.
Live-cell imaging in microfluidic devices now allows the investigation of cellular heterogeneity within microbial populations. In particular, the mother machine technology developed by Wang et al. has been widely employed to investigate single-cell physiological parameters including gene expression, growth rate, mutagenesis, and response to antibiotics. One of the advantages of the mother machine technology is the ability to generate vast amounts of images; however, the time consuming analysis of these images constitutes a severe bottleneck. Here we overcome this limitation by introducing MMHelper ( https://doi.org/10.5281/zenodo.3254394 ), a publicly available custom software implemented in Python which allows the automated analysis of brightfield or phase contrast, and any associated fluorescence, images of bacteria confined in the mother machine. We show that cell data extracted via MMHelper from tens of thousands of individual cells imaged in brightfield are consistent with results obtained via semi-automated image analysis based on ImageJ. Furthermore, we benchmark our software capability in processing phase contrast images from other laboratories against other publicly available software. We demonstrate that MMHelper has over 90% detection efficiency for brightfield and phase contrast images and provides a new open-source platform for the extraction of single-bacterium data, including cell length, area, and fluorescence intensity.
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Lutz T (2019). New optical super-resolution imaging approaches involving DNA nanotechnology.
Abstract:
New optical super-resolution imaging approaches involving DNA nanotechnology
With recent advances in optical super-resolution microscopy, biological structures can be imaged with single-nanometre resolution using visible light. One implementation thereof, DNA-PAINT (Point Accumulation for Imaging in Nano-scale Topography), is based on the highly specific and transient binding of fluorescently labelled oligonucleotides, the "imager strands", to complementary strands with which the targets are labelled, the "docking strands". The imager-docking binding events are detected as fluorescence blinking and can be localised with single-nanometre precision. From the set of localised events a super-resolution image can be assembled. DNA-PAINT has multiple advantages over other imaging methods, e.g. high photon yields resulting in high resolution, a free choice of fluorophores while being effectively free from photobleaching, straightforward implementation on a conventional fluorescence microscope and the possibility of temporally multiplexed and quantitative imaging.
In this thesis, a test sample based on functionalised microspheres is developed, which allows for optimisation of various DNA-PAINT imaging parameters and for the characterisation and testing of new variations and modifications of DNA-PAINT. One such method which was developed for this thesis, Quencher-Exchange-PAINT, facilitates temporally multiplexed imaging, which is based on the sequential exchange of imager strands targeting different docking strands. The exchange step is replaced by addition of competitive quencher-strands, allowing for rapid, low-crosstalk imager exchange even in biological samples with limited diffusion. Additionally, Proximity-Dependent PAINT is introduced, which enables the imaging of the nanoscale distribution of protein pairs by interaction of two proximity probes which activates DNA-PAINT type binding. The technique is demonstrated both on the microsphere assay as well as in biological samples. Finally, approaches for enhancing the signal-to-noise ratio are explored.
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Richards TA, Massana R, Pagliara S, Hall N (2019). Single cell ecology.
Philos Trans R Soc Lond B Biol Sci,
374(1786).
Abstract:
Single cell ecology.
Cells are the building blocks of life, from single-celled microbes through to multi-cellular organisms. To understand a multitude of biological processes we need to understand how cells behave, how they interact with each other and how they respond to their environment. The use of new methodologies is changing the way we study cells allowing us to study them on minute scales and in unprecedented detail. These same methods are allowing researchers to begin to sample the vast diversity of microbes that dominate natural environments. The aim of this special issue is to bring together research and perspectives on the application of new approaches to understand the biological properties of cells, including how they interact with other biological entities. This article is part of a discussion meeting issue 'Single cell ecology'.
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Brandstrup Morrish R, Hermes M, Metz J, Stone N, Pagliara S, Chahwan R, Palombo F (2019). Single cell imaging of nuclear architecture changes.
Frontiers in Immunology,
7, 1-14.
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2018
Smith A, Kaczmar A, Bamford RA, Smith C, Frustaci S, Kovacs-Simon A, O'Neill P, Moore K, Paszkiewicz K, Titball RW, et al (2018). The Culture Environment Influences Both Gene Regulation and Phenotypic Heterogeneity in Escherichia coli.
FRONTIERS IN MICROBIOLOGY,
9 Author URL.
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Lutz T, Clowsley AH, Lin R, Pagliara S, Di Michele L, Soeller C (2018). Versatile multiplexed super-resolution imaging of nanostructures by Quencher-Exchange-PAINT.
Nano Research,
11(12), 6141-6154.
Abstract:
Versatile multiplexed super-resolution imaging of nanostructures by Quencher-Exchange-PAINT
© 2018, the author(s). The optical super-resolution technique DNA-PAINT (Point Accumulation Imaging in Nanoscale Topography) provides a flexible way to achieve imaging of nanoscale structures at ∼10-nanometer resolution. In DNA-PAINT, fluorescently labeled DNA “imager” strands bind transiently and with high specificity to complementary target “docking” strands anchored to the structure of interest. The localization of single binding events enables the assembly of a super-resolution image, and this approach effectively circumvents photobleaching. The solution exchange of imager strands is the basis of Exchange-PAINT, which enables multiplexed imaging that avoids chromatic aberrations. Fluid exchange during imaging typically requires specialized chambers or washes, which can disturb the sample. Additionally, diffusional washout of imager strands is slow in thick samples such as biological tissue slices. Here, we introduce Quencher-Exchange-PAINT—a new approach to Exchange-PAINT in regular open-top imaging chambers—which overcomes the comparatively slow imager strand switching via diffusional imager washout. Quencher-Exchange-PAINT uses “quencher” strands, i.e. oligonucleotides that prevent the imager from binding to the targets, to rapidly reduce unwanted single-stranded imager concentrations to negligible levels, decoupled from the absolute imager concentration. The quencher strands contain an effective dye quencher that reduces the fluorescence of quenched imager strands to negligible levels. We characterized Quencher-Exchange-PAINT when applied to synthetic, cellular, and thick tissue samples. Quencher-Exchange-PAINT opens the way for efficient multiplexed imaging of complex nanostructures, e.g. in thick tissues, without the need for washing steps. [Figure not available: see fulltext.].
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2017
Hodgson AC, Verstreken CM, Fisher CL, Keyser UF, Pagliara S, Chalut KJ (2017). A microfluidic device for characterizing nuclear deformations.
Lab Chip,
17(5), 805-813.
Abstract:
A microfluidic device for characterizing nuclear deformations.
Cell nuclei experience and respond to a wide range of forces, both in vivo and in vitro. In order to characterize the nuclear response to physical stress, we developed a microfluidic chip and used it to apply mechanical stress to live cells and measure their nuclear deformability. The device design is optimized for the detection of both nucleus and cytoplasm, which can then be conveniently quantified using a custom-written Matlab program. We measured nuclear sizes and strains of embryonic stem cells, for which we observed negative Poisson ratios in the nuclei. In addition, we were able to detect changes in the nuclear response after treatment with actin depolymerizing and chromatin decondensing agents. Finally, we showed that the device can be used for biologically relevant high-resolution confocal imaging of cells under compression. Thus, the device presented here allows for accurate physical phenotyping at high throughput and has the potential to be applied to a range of cell types.
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Bamford RA, Smith A, Metz J, Glover G, Titball RW, Pagliara S (2017). Investigating the physiology of viable but non-culturable bacteria by microfluidics and time-lapse microscopy.
BMC Biology,
15(1).
Abstract:
Investigating the physiology of viable but non-culturable bacteria by microfluidics and time-lapse microscopy
© 2017 Pagliara et al. Background: Clonal microbial populations often harbor rare phenotypic variants that are typically hidden within the majority of the remaining cells, but are crucial for the population's resilience to external perturbations. Persister and viable but non-culturable (VBNC) cells are two important clonal bacterial subpopulations that can survive antibiotic treatment. Both persister and VBNC cells pose a serious threat to human health. However, unlike persister cells, which quickly resume growth following drug removal, VBNC cells can remain non-growing for prolonged periods of time, thus eluding detection via traditional microbiological assays. Therefore, understanding the molecular mechanisms underlying the formation of VBNC cells requires the characterization of the clonal population with single-cell resolution. A combination of microfluidics, time-lapse microscopy, and fluorescent reporter strains offers the perfect platform for investigating individual cells while manipulating their environment. Methods: Here, we report a novel single-cell approach to investigate VBNC cells. We perform drug treatment, bacterial culturing, and live/dead staining in series by using transcriptional reporter strains and novel adaptations to the mother machine technology. Since we track each cell throughout the experiment, we are able to quantify the size, morphology and fluorescence that each VBNC cell displayed before, during and after drug treatment. Results: We show that VBNC cells are not dead or dying cells but share similar phenotypic features with persister cells, suggesting a link between these two subpopulations, at least in the Escherichia coli strain under investigation. We strengthen this link by demonstrating that, before drug treatment, both persister and VBNC cells can be distinguished from the remainder of the population by their lower fluorescence when using a reporter strain for tnaC, encoding the leader peptide of the tnaCAB operon responsible for tryptophan metabolism. Conclusion: Our data demonstrates the suitability of our approach for studying the physiology of non-growing cells in response to external perturbations. Our approach will allow the identification of novel biomarkers for the isolation of VBNC and persister cells and will open new opportunities to map the detailed biochemical makeup of these clonal subpopulations.
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2016
Cama J, Schaich M, Al Nahas K, Hernández-Ainsa S, Pagliara S, Keyser UF (2016). Direct Optofluidic Measurement of the Lipid Permeability of Fluoroquinolones.
Sci Rep,
6Abstract:
Direct Optofluidic Measurement of the Lipid Permeability of Fluoroquinolones.
Quantifying drug permeability across lipid membranes is crucial for drug development. In addition, reduced membrane permeability is a leading cause of antibiotic resistance in bacteria, and hence there is a need for new technologies that can quantify antibiotic transport across biological membranes. We recently developed an optofluidic assay that directly determines the permeability coefficient of autofluorescent drug molecules across lipid membranes. Using ultraviolet fluorescence microscopy, we directly track drug accumulation in giant lipid vesicles as they traverse a microfluidic device while exposed to the drug. Importantly, our measurement does not require the knowledge of the octanol partition coefficient of the drug - we directly determine the permeability coefficient for the specific drug-lipid system. In this work, we report measurements on a range of fluoroquinolone antibiotics and find that their pH dependent lipid permeability can span over two orders of magnitude. We describe various technical improvements for our assay, and provide a new graphical user interface for data analysis to make the technology easier to use for the wider community.
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Locatelli E, Pierno M, Baldovin F, Orlandini E, Tan Y, Pagliara S (2016). Single-File Escape of Colloidal Particles from Microfluidic Channels.
Physical Review Letters,
117(3).
Abstract:
Single-File Escape of Colloidal Particles from Microfluidic Channels
© 2016 American Physical Society. Single-file diffusion is a ubiquitous physical process exploited by living and synthetic systems to exchange molecules with their environment. It is paramount to quantify the escape time needed for single files of particles to exit from constraining synthetic channels and biological pores. This quantity depends on complex cooperative effects, whose predominance can only be established through a strict comparison between theory and experiments. By using colloidal particles, optical manipulation, microfluidics, digital microscopy, and theoretical analysis we uncover the self-similar character of the escape process and provide closed-formula evaluations of the escape time. We find that the escape time scales inversely with the diffusion coefficient of the last particle to leave the channel. Importantly, we find that at the investigated microscale, bias forces as tiny as 10-15 N determine the magnitude of the escape time by drastically reducing interparticle collisions. Our findings provide crucial guidelines to optimize the design of micro- and nanodevices for a variety of applications including drug delivery, particle filtering, and transport in geometrical constrictions.
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2015
Cama J, Bajaj H, Pagliara S, Maier T, Braun Y, Winterhalter M, Keyser UF (2015). Quantification of Fluoroquinolone Uptake through the Outer Membrane Channel OmpF of Escherichia coli.
J Am Chem Soc,
137(43), 13836-13843.
Abstract:
Quantification of Fluoroquinolone Uptake through the Outer Membrane Channel OmpF of Escherichia coli.
Decreased drug accumulation is a common cause of antibiotic resistance in microorganisms. However, there are few reliable general techniques capable of quantifying drug uptake through bacterial membranes. We present a semiquantitative optofluidic assay for studying the uptake of autofluorescent drug molecules in single liposomes. We studied the effect of the Escherichia coli outer membrane channel OmpF on the accumulation of the fluoroquinolone antibiotic, norfloxacin, in proteoliposomes. Measurements were performed at pH 5 and pH 7, corresponding to two different charge states of norfloxacin that bacteria are likely to encounter in the human gastrointestinal tract. At both pH values, the porins significantly enhance drug permeation across the proteoliposome membranes. At pH 5, where norfloxacin permeability across pure phospholipid membranes is low, the porins increase drug permeability by 50-fold on average. We estimate a flux of about 10 norfloxacin molecules per second per OmpF trimer in the presence of a 1 mM concentration gradient of norfloxacin. We also performed single channel electrophysiology measurements and found that the application of transmembrane voltages causes an electric field driven uptake in addition to concentration driven diffusion. We use our results to propose a physical mechanism for the pH mediated change in bacterial susceptibility to fluoroquinolone antibiotics.
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Otto O, Rosendahl P, Mietke A, Golfier S, Herold C, Klaue D, Girardo S, Pagliara S, Ekpenyong A, Jacobi A, et al (2015). Real-time deformability cytometry: On-the-fly cell mechanical phenotyping.
Nature Methods,
12(3), 199-202.
Abstract:
Real-time deformability cytometry: On-the-fly cell mechanical phenotyping
© 2015 Nature America, Inc. We introduce real-time deformability cytometry (RT-DC) for continuous cell mechanical characterization of large populations (>100,000 cells) with analysis rates greater than 100 cells/s. RT-DC is sensitive to cytoskeletal alterations and can distinguish cell-cycle phases, track stem cell differentiation into distinct lineages and identify cell populations in whole blood by their mechanical fingerprints. This technique adds a new marker-free dimension to flow cytometry with diverse applications in biology, biotechnology and medicine.
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2014
Cama J, Chimerel C, Pagliara S, Javer A, Keyser UF (2014). A label-free microfluidic assay to quantitatively study antibiotic diffusion through lipid membranes.
Lab Chip,
14(13), 2303-2308.
Abstract:
A label-free microfluidic assay to quantitatively study antibiotic diffusion through lipid membranes.
With the rise in antibiotic resistance amongst pathogenic bacteria, the study of antibiotic activity and transport across cell membranes is gaining widespread importance. We present a novel, label-free microfluidic assay that quantifies the permeability coefficient of a broad spectrum fluoroquinolone antibiotic, norfloxacin, across lipid membranes using the UV autofluorescence of the drug. We use giant lipid vesicles as highly controlled model systems to study the diffusion through lipid membranes. Our technique directly determines the permeability coefficient without requiring the measurement of the partition coefficient of the antibiotic.
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Pagliara S, Franze K, McClain CR, Wylde GW, Fisher CL, Franklin RJM, Kabla AJ, Keyser UF, Chalut KJ (2014). Auxetic nuclei in embryonic stem cells exiting pluripotency.
Nature Materials,
13(6), 638-644.
Abstract:
Auxetic nuclei in embryonic stem cells exiting pluripotency
Embryonic stem cells (ESCs) self-renew in a state of naïve pluripotency in which they are competent to generate all somatic cells. It has been hypothesized that, before irreversibly committing, ESCs pass through at least one metastable transition state. This transition would represent a gateway for differentiation and reprogramming of somatic cells. Here, we show that during the transition, the nuclei of ESCs are auxetic: they exhibit a cross-sectional expansion when stretched and a cross-sectional contraction when compressed, and their stiffness increases under compression. We also show that the auxetic phenotype of transition ESC nuclei is driven at least in part by global chromatin decondensation. Through the regulation of molecular turnover in the differentiating nucleus by external forces, auxeticity could be a key element in mechanotransduction. Our findings highlight the importance of nuclear structure in the regulation of differentiation and reprogramming. © 2014 Macmillan Publishers Limited.
Abstract.
Pagliara S, Dettmer SL, Keyser UF (2014). Channel-facilitated diffusion boosted by particle binding at the channel entrance.
Physical Review Letters,
113(4).
Abstract:
Channel-facilitated diffusion boosted by particle binding at the channel entrance
We investigate single-file diffusion of Brownian particles in arrays of closely confining microchannels permeated by a variety of attractive optical potentials and connecting two baths with equal particle concentration. We simultaneously test free diffusion in the channel, diffusion in optical traps coupled in the center of the channel, and diffusion in traps extending into the baths. We found that both classes of attractive optical potentials enhance the translocation rate through the channel with respect to free diffusion. Surprisingly, for the latter class of potentials we measure a 40-fold enhancement in the translocation rate with respect to free diffusion and find a sublinear power law dependence of the translocation rate on the average number of particles in the channel. Our results reveal the function of particle binding at the channel entrances for diffusive transport and open the way to a better understanding of membrane transport and design of synthetic membranes with enhanced diffusion rate. © 2014 American Physical Society.
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Pagliara S, Dettmer SL, Misiunas K, Lea L, Tan Y, Keyser UF (2014). Diffusion coefficients and particle transport in synthetic membrane channels.
European Physical Journal: Special Topics,
223(14), 3145-3163.
Abstract:
Diffusion coefficients and particle transport in synthetic membrane channels
© 2014, EDP Sciences and Springer. Diffusion in constrained geometries is paramount to transport across biological membranes and in mesoporous materials. Although the transported species vary from system to system, the underlying physical mechanisms are universal. However, there is an imbalance between theory and quantitative experimental model systems. We have recently introduced a new synthetic approach to mimic molecular diffusion based on colloidal particles, digital video microscopy, particle tracking, microfluidics and holographic optical tweezers. In this paper we report useful guidelines for the fabrication, handling and characterisation of the microfluidic chips and a study of diffusion coefficients, particle attempt and translocation rates through microfluidic channels with cross sections of different dimensions.
Abstract.
Dettmer SL, Keyser UF, Pagliara S (2014). Local characterization of hindered Brownian motion by using digital video microscopy and 3D particle tracking.
Rev Sci Instrum,
85(2).
Abstract:
Local characterization of hindered Brownian motion by using digital video microscopy and 3D particle tracking.
In this article we present methods for measuring hindered Brownian motion in the confinement of complex 3D geometries using digital video microscopy. Here we discuss essential features of automated 3D particle tracking as well as diffusion data analysis. By introducing local mean squared displacement-vs-time curves, we are able to simultaneously measure the spatial dependence of diffusion coefficients, tracking accuracies and drift velocities. Such local measurements allow a more detailed and appropriate description of strongly heterogeneous systems as opposed to global measurements. Finite size effects of the tracking region on measuring mean squared displacements are also discussed. The use of these methods was crucial for the measurement of the diffusive behavior of spherical polystyrene particles (505 nm diameter) in a microfluidic chip. The particles explored an array of parallel channels with different cross sections as well as the bulk reservoirs. For this experiment we present the measurement of local tracking accuracies in all three axial directions as well as the diffusivity parallel to the channel axis while we observed no significant flow but purely Brownian motion. Finally, the presented algorithm is suitable also for tracking of fluorescently labeled particles and particles driven by an external force, e.g. electrokinetic or dielectrophoretic forces.
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Schleicher KD, Dettmer SL, Kapinos LE, Pagliara S, Keyser UF, Jeney S, Lim RYH (2014). Selective transport control on molecular velcro made from intrinsically disordered proteins.
Nature Nanotechnology,
9(7), 525-530.
Abstract:
Selective transport control on molecular velcro made from intrinsically disordered proteins
The selectivity and speed of many biological transport processes transpire from a 'reduction of dimensionality' that confines diffusion to one or two dimensions instead of three. This behaviour remains highly sought after on polymeric surfaces as a means to expedite diffusional search processes in molecular engineered systems. Here, we have reconstituted the two-dimensional diffusion of colloidal particles on a molecular brush surface. The surface is composed of phenylalanine-glycine nucleoporins (FG Nups) - intrinsically disordered proteins that facilitate selective transport through nuclear pore complexes in eukaryotic cells. Local and ensemble-level experiments involving optical trapping using a photonic force microscope and particle tracking by video microscopy, respectively, reveal that 1-μm-sized colloidal particles bearing nuclear transport receptors called karyopherins can exhibit behaviour that varies from highly localized to unhindered two-dimensional diffusion. Particle diffusivity is controlled by varying the amount of free karyopherins in solution, which modulates the multivalency of Kap-binding sites within the molecular brush. We conclude that the FG Nups resemble stimuli-responsive molecular 'velcro', which can impart 'reduction of dimensionality' as a means of biomimetic transport control in artificial environments. © 2014 Macmillan Publishers Limited.
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2013
Camposeo A, Greenfeld I, Tantussi F, Pagliara S, Moffa M, Fuso F, Allegrini M, Zussman E, Pisignano D (2013). Local mechanical properties of electrospun fibers correlate to their internal nanostructure.
Nano Lett,
13(11), 5056-5062.
Abstract:
Local mechanical properties of electrospun fibers correlate to their internal nanostructure.
The properties of polymeric nanofibers can be tailored and enhanced by properly managing the structure of the polymer molecules at the nanoscale. Although electrospun polymer fibers are increasingly exploited in many technological applications, their internal nanostructure, determining their improved physical properties, is still poorly investigated and understood. Here, we unravel the internal structure of electrospun functional nanofibers made by prototype conjugated polymers. The unique features of near-field optical measurements are exploited to investigate the nanoscale spatial variation of the polymer density, evidencing the presence of a dense internal core embedded in a less dense polymeric shell. Interestingly, nanoscale mapping the fiber Young's modulus demonstrates that the dense core is stiffer than the polymeric, less dense shell. These findings are rationalized by developing a theoretical model and simulations of the polymer molecular structural evolution during the electrospinning process. This model predicts that the stretching of the polymer network induces a contraction of the network toward the jet center with a local increase of the polymer density, as observed in the solid structure. The found complex internal structure opens an interesting perspective for improving and tailoring the molecular morphology and multifunctional electronic and optical properties of polymer fibers.
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Pagliara S, Schwall C, Keyser UF (2013). Optimizing diffusive transport through a synthetic membrane channel.
Adv Mater,
25(6), 844-849.
Author URL.
2012
Pagliara S, Polini A, Camposeo A, Schröder HC, Müller WEG, Pisignano D (2012). Electrical properties of in vitro biomineralized recombinant silicatein deposited by microfluidics.
Applied Physics Letters,
101(19).
Abstract:
Electrical properties of in vitro biomineralized recombinant silicatein deposited by microfluidics
We report the fabrication of silica dielectrics obtained by in vitro biomineralization of recombinant silicatein. We exploit pressure-driven microfluidics to deposit silicatein which catalyses the deposition of silica features with thickness in the range 2-6 μm. We follow the biomineralization process with staining and confocal fluorescence for an incubation time up to 5 days and correspondingly characterize the leakage current through the resulting biomineralized silica layer by embedding it into a metal-insulator-metal device. We further characterize the morphology of the biosilica surface through atomic force and scanning electron microscopy and demonstrate the electrical insulation within planar electrodes patterned over such surface with leakage currents in the pA range for applied bias up to tens of V. © 2012 American Institute of Physics.
Abstract.
Foderá V, Pagliara S, Otto O, Keyser UF, Donald AM (2012). Microfluidics reveals a flow-induced large-scale polymorphism of protein aggregates.
Journal of Physical Chemistry Letters,
3(19), 2803-2807.
Abstract:
Microfluidics reveals a flow-induced large-scale polymorphism of protein aggregates
Amyloid fibrils are characterized by a structural arrangement of cross β-sheet as a common motif. However they can also experience a more complicated packing into a variety of 3D supramolecular structures (polymorphism). Confinement and flow rate play a crucial role in protein aggregation in living systems, but controlling such parameters during in vitro experiments still remains an unsolved problem. Here we present evidence of the effect of flow rate on the aggregation process in a confined environment using microfluidics. Specifically, we show that a gradual transition from spherical aggregates, that is, spherulites, to thick fiber-like structures takes place as a result of increasing the flow rate. Such results have implications both for a basic understanding of the mechanism behind aggregation phenomena and in the development of novel biomaterials. © 2012 American Chemical Society.
Abstract.
Polini A, Pagliara S, Camposeo A, Cingolani R, Wang X, Schröder HC, Müller WEG, Pisignano D (2012). Optical properties of in-vitro biomineralised silica.
Sci Rep,
2Abstract:
Optical properties of in-vitro biomineralised silica.
Silicon is the second most common element on the Earth's crust and its oxide (SiO(2)) the most abundant mineral. Silica and silicates are widely used in medicine and industry as well as in micro- and nano-optics and electronics. However, the fabrication of glass fibres and components requires high temperature and non-physiological conditions, in contrast to biosilica structures in animals and plants. Here, we show for the first time the use of recombinant silicatein-α, the most abundant subunit of sponge proteins catalyzing biosilicification reactions, to direct the formation of optical waveguides in-vitro through soft microlithography. The artificial biosilica fibres mimic the natural sponge spicules, exhibiting refractive index values suitable for confinement of light within waveguides, with optical losses in the range of 5-10 cm(-1), suitable for application in lab-on-chips systems. This method extends biosilicification to the controlled fabrication of optical components by physiological processing conditions, hardly addressed by conventional technologies.
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Lee CW, Pagliara S, Keyser U, Baumberg JJ (2012). Perpendicular coupling to in-plane photonics using arc waveguides fabricated via two-photon polymerization.
Applied Physics Letters,
100(17).
Abstract:
Perpendicular coupling to in-plane photonics using arc waveguides fabricated via two-photon polymerization
We demonstrate the concept of vertically standing arc waveguides to couple normally incident light into the plane of a photonic circuit or sensor array. The simple one-step direct write fabrication uses a low power picosecond microchip laser for two-photon polymerization with high-speed and low-cost. Arc waveguides with different arc radii and waveguide port diameters are obtained, with insertion loss down to 1.5 dB. This demonstration of a distinctly different architecture employing unsupported arc waveguides adds another dimension to photonic integration and opens up applications for environmental sensors, integrated microfluidics, bio-assay chips, as well as offering an alternate way of input/output-coupling to planar waveguides. © 2012 American Institute of Physics.
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Ekpenyong AE, Whyte G, Chalut K, Pagliara S, Lautenschläger F, Fiddler C, Paschke S, Keyser UF, Chilvers ER, Guck J, et al (2012). Viscoelastic properties of differentiating blood cells are fate- and function-dependent.
PLoS One,
7(9).
Abstract:
Viscoelastic properties of differentiating blood cells are fate- and function-dependent.
Although cellular mechanical properties are known to alter during stem cell differentiation, understanding of the functional relevance of such alterations is incomplete. Here, we show that during the course of differentiation of human myeloid precursor cells into three different lineages, the cells alter their viscoelastic properties, measured using an optical stretcher, to suit their ultimate fate and function. Myeloid cells circulating in blood have to be advected through constrictions in blood vessels, engendering the need for compliance at short time-scales (minutes), compared to undifferentiated cells. These findings suggest that reduction in steady-state viscosity is a physiological adaptation for enhanced migration through tissues. Our results indicate that the material properties of cells define their function, can be used as a cell differentiation marker and could serve as target for novel therapies.
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2011
Tu D, Pagliara S, Cingolani R, Pisignano D (2011). An electrospun fiber phototransistor by the conjugated polymer poly[2-methoxy-5-(2'-ethylhexyloxy)-1,4-phenylene-vinylene].
Applied Physics Letters,
98(2).
Abstract:
An electrospun fiber phototransistor by the conjugated polymer poly[2-methoxy-5-(2'-ethylhexyloxy)-1,4-phenylene-vinylene]
We investigate the photoresponse of field-effect transistors based on conjugated polymer electrospun fibers. The electrical performances of single fiber transistors are controlled by modulating the channel conductivity under white light illumination. We demonstrate a photoresponsivity up to 100 mA/W for a 500-nm channel width fiber phototransistor illuminated by an intensity of 9.6 mW/cm2. Studying the photoresponse switching cycles evidences that the photocurrent relaxation time can be reduced down to about 40 s by increasing the fiber surface-to-volume ratio. © 2011 American Institute of Physics.
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Polini A, Pagliara S, Camposeo A, Biasco A, Schröder HC, Müller WEG, Pisignano D (2011). Biosilica electrically-insulating layers by soft lithography-assisted biomineralisation with recombinant silicatein.
Adv Mater,
23(40), 4674-4678.
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Pagliara S, Chimerel C, Aarts DGAL, Langford R, Keyser UF (2011). Colloid flow control in microchannels and detection by laser scattering.
Progress in Colloid and Polymer Science,
139, 45-49.
Abstract:
Colloid flow control in microchannels and detection by laser scattering
We introduce a new approach towards the flow control and detection of colloids in microfluidic specimens. We fabricate hybrid polydimethylsiloxane (PDMS)/glass microfluidic chips equipped with parallel micrometer and sub-micrometer channels with different width and thickness. We image and detect the colloid flow direction through the microchannels by coupling laser-light-scattering in a restricted region of a single channel. We control single polymer colloids by means of a computerized pressure-based flow control system and study the Poiseuille flow through channels with different square cross section. We demonstrate the possibility of in situ sensing populations of colloids with different dimensions down to the sub-100 nm scale. © Springer-Verlag Berlin Heidelberg 2012.
Abstract.
Fodera V, Pagliara S, Keyser UF, Donald AM (2011). Flow-induced polymorphism of protein aggregates in micro channels.
Author URL.
Pagliara S, Vitiello MS, Camposeo A, Polini A, Cingolani R, Scamarcio G, Pisignano D (2011). Optical anisotropy in single light-emitting polymer nanofibers.
Journal of Physical Chemistry C,
115(42), 20399-20405.
Abstract:
Optical anisotropy in single light-emitting polymer nanofibers
We investigate the optical anisotropy of single nanofibers realized by electrospinning a conjugated polymer. Polarized infrared and micro-Raman measurements evidence a higher degree of molecular orientation in fibers processed from tetrahydrofuran solutions, with respect to samples spun from mixture with dimethyl sulfoxide. The fraction of ordered molecules is correlated to the fibers morphology. Polarized photoluminescence highlights a larger red shift (60 meV) for spectra from fibers processed from tetrahydrofuran, confirming the higher achieved molecular order resulting in reduced interchain separation and hence excitonic emission with lower transition energies compared to that from randomly aligned molecules. Conjugated polymer fibers are obtained, emitting light with polarization ratios up to 5, usable as polarized photonic nanosources. © 2011 American Chemical Society.
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Pagliara S, Chimerel C, Langford R, Aarts DGAL, Keyser UF (2011). Parallel sub-micrometre channels with different dimensions for laser scattering detection.
Lab Chip,
11(19), 3365-3368.
Abstract:
Parallel sub-micrometre channels with different dimensions for laser scattering detection.
A novel and simple approach for the realization of polymer sub-micrometre channels is introduced by exploiting replica molding of Pt wires deposited by focused ion beam. We fabricate arrays of parallel channels with typical dimensions down to 600 nm and with variable height. We characterize the pressure-driven transport of polymer colloids through the channels in terms of the translocation frequency, amplitude and duration by implementing a laser scattering detection technique. We propose a prototype application of the presented platform such as the in situ sizing and sensing of populations of particles with different dimensions down to 50 nm.
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Tu D, Pagliara S, Camposeo A, Potente G, Mele E, Cingolani R, Pisignano D (2011). Soft nanolithography by polymer fibers.
Advanced Functional Materials,
21(6), 1140-1145.
Abstract:
Soft nanolithography by polymer fibers
We report on the use of polymer fibers for large-area soft nanolithography on organic and inorganic surfaces with 50 nm resolution. The morphology of fibers and of the corresponding patterned gap is investigated, demonstrating a lateral dimension downscaling of up to nine times, which greatly increases the achieved resolution during pattern transfer. In this way, we realize polymer field effect transistors with channel length and width as low as 250 nm that are expected to show transistor transition frequency up to a few MHz, and are thus exploitable as low-cost radio-frequency identification devices. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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2010
Polini A, Pagliara S, Stabile R, Netti GS, Roca L, Prattichizzo C, Gesualdo L, Cingolani R, Pisignano D (2010). Collagen-functionalised electrospun polymer fibers for bioengineering applications.
Soft Matter,
6(8), 1668-1674.
Abstract:
Collagen-functionalised electrospun polymer fibers for bioengineering applications
Polymer electrospun fibers are gaining increasing importance in nanobiotechnology, due to their intrinsic three-dimensional topography and biochemical flexibility. Here we present an in-depth study of protein functionalisation for polymethylmethacrylate fibers. We compare different coating approaches for type I collagen, including physisorption and covalent binding methods relying on functional linkers. The biofunctionalised fibers are investigated by scanning electron and confocal laser scanning microscopy, wettability measurements, Fourier-transform infrared spectroscopy, and protein quantification assays. We demonstrate that the largest amount of proteins adsorbed on fibers does not determine the best performance in terms of cell attachment and proliferation in vitro, which is instead related to the type of linking and the relevant role played by adsorption of serum biomolecules on the three-dimensional nanostructures. This study is relevant for designing and engineering novel biomaterials and scaffold architectures based on electrospun nanofibers. © 2010 the Royal Society of Chemistry.
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Camposeo A, Pagliara S, Polini A, Pisignano D (2010). Conjugated polymer nanofibers: Novel light sources for microfluidic systems.
Optics InfoBase Conference PapersAbstract:
Conjugated polymer nanofibers: Novel light sources for microfluidic systems
We report on the fabrication of electrospun light-emitting polymer nanofibers made by conjugated and dye-doped polymers. The fibers exhibit diameter down to a few tens of nm, and emission in the visible and near-infrared spectral range. The investigation of the photoluminescence properties of the fibers evidences an emission mainly polarized along the fiber axis. The fibers are integrated in prototype microfluidic system as micro-scale polarized light sources. © 2009 Optical Society of America.
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Pagliara S, Camposeo A, Mele E, Persano L, Cingolani R, Pisignano D (2010). Enhancement of light polarization from electrospun polymer fibers by room temperature nanoimprint lithography.
Nanotechnology,
21(21).
Abstract:
Enhancement of light polarization from electrospun polymer fibers by room temperature nanoimprint lithography.
We demonstrate the enhancement of the polarization ratio of light emitted from electrospun conjugated polymer nanofibers, by means of nanoimprint lithography carried out at room temperature. We provide evidence of tailoring the polarization properties by patterning light-emitting fibers at the nanoscale. The polarization ratios are increased up to a factor of 2.4 by gratings with periodicity (560 nm) matching the emission wavelength of the employed conjugated polymer. The use of room temperature nanoimprint lithography to pattern light-emitting polymer nanofibers represents a strategic route for realizing photonic crystals and distributed feedback polarized emitters on one-dimensional organic nanostructures.
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Pagliara S, Camposeo A, Neves AAR, Polini A, Cingolani R, Pisignano D (2010). Integration of polarized light-emitting nanostructures for biomarker sensing. European Cells and Materials, 20(SUPPL.3).
Neves AAR, Camposeo A, Pagliara S, Saija R, Borghese F, Denti P, Iatì MA, Cingolani R, Maragò OM, Pisignano D, et al (2010). Rotational dynamics of optically trapped nanofibers.
Opt Express,
18(2), 822-830.
Abstract:
Rotational dynamics of optically trapped nanofibers.
We report on the experimental evidence of tilted polymer nanofiber rotation, using a highly focused linear polarized Gaussian beam. Torque is controlled by varying trapping power or fiber tilt angle. This suggests an alternative strategy to previously reported approaches for the rotation of nano-objects, to test fundamental theoretical aspects. We compare experimental rotation frequencies to calculations based on T-Matrix formalism, which accurately reproduces measured data, thus providing a comprehensive description of trapping and rotation dynamics of the linear nanostructures.
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Tu D, Pagliara S, Camposeo A, Persano L, Cingolani R, Pisignano D (2010). Single light-emitting polymer nanofiber field-effect transistors.
Nanoscale,
2(10), 2217-2222.
Abstract:
Single light-emitting polymer nanofiber field-effect transistors.
We report on single nanofiber field-effect transistors made by the light-emitting polymer, poly(2-methoxy-5-(2-ethylhexoxy)-1,4-phenylenevinylene). We measure electrical performances comparable to or better than those of thin-film transistors by the same organic semiconductor, due to the molecular alignment induced by electrospinning, such as hole mobility of the order of 10(-3) cm(2) V(-1) s(-1) and on/off current ratios up to 780. In addition, we observe controllable photoluminescence intensity quenching by varying the gate voltage up to -40 V with device operation in the luministor mode. Single light-emitting polymer nanofiber transistors coupling electrical and optical functionalities open the way towards low cost and flexible one-dimensional switches and nanofiber-based light-emitting transistors.
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Pagliara S, Camposeo A, Di Benedetto F, Polini A, Mele E, Persano L, Cingolani R, Pisignano D (2010). Study of optical properties of electrospun light-emitting polymer fibers.
Superlattices and Microstructures,
47(1), 145-149.
Abstract:
Study of optical properties of electrospun light-emitting polymer fibers
We realize light-emitting polymer fibers based on both optically inert polymers doped by molecules exhibiting optical gain and optically active conjugated polymers. Waveguiding properties of the produced polymer structures are demonstrated, with a loss coefficient of around 103 cm-1. We also find that single polymer fibers doped with gain molecules form Fabry-Pérot cavities, showing photoluminescence spectra with modes equally spaced by 1.7 nm. Coherent emission is demonstrated from fibers made upon increasing the excitation fluence above threshold values of the order of a few tens of μJ/cm2. © 2009 Elsevier Ltd. All rights reserved.
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2009
Pagliara S, Camposeo A, Polini A, Cingolani R, Pisignano D (2009). Electrospun light-emitting nanofibers as excitation source in microfluidic devices.
Lab Chip,
9(19), 2851-2856.
Abstract:
Electrospun light-emitting nanofibers as excitation source in microfluidic devices.
We introduce the integration of organic, polarised light-emitting electrospun nanofibers and lab-on-a-chip microchannel geometries. The alignment and spinning electric field leads to ordered mesoscopic active areas, up to many mm(2), which exhibit polarised light emission and are fully compatible with microlithographies and microfluidics. We utilise the nanofibers demonstrating the photo-excitation of flowing dye chromophores in microchannels. This leads to easy decoupling the excitation and sample emission by polarisation analysers, thus remarkably increasing the imaging signal to background noise ratio.
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Pagliara S, Camposeo A, Cingolani R, Pisignano D (2009). Hierarchical assembly of light-emitting polymer nanofibers in helical morphologies.
Applied Physics Letters,
95(26).
Abstract:
Hierarchical assembly of light-emitting polymer nanofibers in helical morphologies
Single electrospun nanofibers of light-emitting conjugated polymers hierarchically assemble at nano- to macroscopic lengthscales in various helical morphologies. At nanoscopic lengthscales, molecular chains follow the microscopic assembly, prevalently aligning along the fiber dynamic axis, as demonstrated by polarized photoluminescence spectroscopy. The role of molecular weight in the resulting assembling and optical properties is highlighted and discussed. Nanofibers based on the heaviest polymer exhibit the most stretched helical geometries and the highest suppression of the excitonic energy migration, resulting in the most blue-shifted photoluminescence with respect to thin films. © 2009 American Institute of Physics.
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2008
Di Benedetto F, Camposeo A, Pagliara S, Mele E, Persano L, Stabile R, Cingolani R, Pisignano D (2008). Patterning of light-emitting conjugated polymer nanofibres.
Nat Nanotechnol,
3(10), 614-619.
Abstract:
Patterning of light-emitting conjugated polymer nanofibres.
Organic materials have revolutionized optoelectronics by their processability, flexibility and low cost, with application to light-emitting devices for full-colour screens, solar cells and lasers. Some low-dimensional organic semiconductor structures exhibit properties resembling those of inorganics, such as polarized emission and enhanced electroluminescence. One-dimensional metallic, III-V and II-VI nanostructures have also been the subject of intense investigation as building blocks for nanoelectronics and photonics. Given that one-dimensional polymer nanostructures, such as polymer nanofibres, are compatible with sub-micrometre patterning capability and electromagnetic confinement within subwavelength volumes, they can offer the benefits of organic light sources to nanoscale optics. Here we report on the optical properties of fully conjugated, electrospun polymer nanofibres. We assess their waveguiding performance and emission tuneability in the whole visible range. We demonstrate the enhancement of the fibre forward emission through imprinting periodic nanostructures using room-temperature nanoimprint lithography, and investigate the angular dispersion of differently polarized emitted light.
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2007
Pagliara S, Persano L, Camposeo A, Cingolani R, Pisignano D (2007). Registration accuracy in multilevel soft lithography.
NANOTECHNOLOGY,
18(17).
Author URL.
Pagliara S, Persano L, Camposeo A, Cingolani R, Pisignano D (2007). Registration accuracy in multilevel soft lithography.
Nanotechnology,
18(17).
Abstract:
Registration accuracy in multilevel soft lithography
We investigate the registration accuracy achievable by multilevel soft lithography. By a specifically designed soft lithography aligner, we obtain, for the average misalignment between two registered patterned organic layers, values decreasing from (4.96 ± 0.02) to (0.50 ± 0.01) μm upon increasing the Young's modulus of the stamp materials from 1.8 to 2600 MPa. This clearly identifies in the stamp distortions the main factor limiting the registration accuracy. The potentiality to achieve registration within 500 nm over areas of 50 × 50 μm2 is demonstrated, opening the way for soft lithographies with high overlay alignment accuracy. © IOP Publishing Ltd.
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