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The actuation of micro- and nanostructures controlled by external stimuli remains one of the exciting challenges in nanotechnology due to the wealth of fundamental questions and potential applications in energy harvesting, robotics, sensing, biomedicine, and tunable metamaterials. Photoactuation utilizes the conversion of light into motion through reversible chemical and physical processes and enables remote and spatiotemporal control of the actuation. Here, we report a fast light-to-motion conversion in few-nanometer thick bare polydopamine (PDA) membranes stimulated by visible light. Light-induced heating of PDA leads to desorption of water molecules and contraction of membranes in less than 140 µs. Switching off the light leads to a spontaneous expansion in less than 20 ms due to heat dissipation and water adsorption. Our findings demonstrate that pristine PDA membranes are multiresponsive materials that can be harnessed as robust building blocks for soft, micro-, and nanoscale actuators stimulated by light, temperature, and moisture level.
Assuntos
Nanoestruturas , Polímeros , Indóis , Nanotecnologia , Polímeros/químicaRESUMO
Measurement of the nitric oxide (NO) concentration in living cells in the physiological nanomolar range is crucial in understanding NO biochemical functions, as well as in characterizing the efficiency and kinetics of NO delivery by NO-releasing drugs. Here, we show that fluorescence correlation spectroscopy (FCS) is perfectly suited for these purposes, due to its sensitivity, selectivity, and spatial resolution. Using the fluorescent indicators, diaminofluoresceins (DAFs), and FCS, we measured the NO concentrations in NO-producing living human primary endothelial cells, as well as NO delivery kinetics, by an external NO donor to the immortal human epithelial living cells. Due to the high spatial resolution of FCS, the NO concentration in different parts of the cells were also measured. The detection of nitric oxide by means of diaminofluoresceins is much more efficient and faster in living cells than in PBS solutions, even though the conversion to the fluorescent form is a multi-step reaction.
Assuntos
Óxido Nítrico/análise , Espectrometria de Fluorescência/métodos , Sobrevivência Celular , Corantes Fluorescentes/análise , Células HeLa , Células Endoteliais da Veia Umbilical Humana , Humanos , Imagem ÓpticaRESUMO
Bacterial DNA gyrase introduces negative supercoils into chromosomal DNA and relaxes positive supercoils introduced by replication and transiently by transcription. Removal of these positive supercoils is essential for replication fork progression and for the overall unlinking of the two duplex DNA strands, as well as for ongoing transcription. To address how gyrase copes with these topological challenges, we used high-speed single-molecule fluorescence imaging in live Escherichia coli cells. We demonstrate that at least 300 gyrase molecules are stably bound to the chromosome at any time, with â¼12 enzymes enriched near each replication fork. Trapping of reaction intermediates with ciprofloxacin revealed complexes undergoing catalysis. Dwell times of â¼2 s were observed for the dispersed gyrase molecules, which we propose maintain steady-state levels of negative supercoiling of the chromosome. In contrast, the dwell time of replisome-proximal molecules was â¼8 s, consistent with these catalyzing processive positive supercoil relaxation in front of the progressing replisome.
Assuntos
DNA Girase/química , DNA Super-Helicoidal/química , Proteínas de Ligação a DNA/química , Escherichia coli/enzimologia , Catálise , DNA Girase/genética , DNA Girase/isolamento & purificação , DNA Super-Helicoidal/genética , DNA Super-Helicoidal/isolamento & purificação , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/isolamento & purificação , Ligação Proteica , Imagem Individual de MoléculaRESUMO
Fluorescent submicron particles of fluorinated methacrylate (HFMBA) with long-term stability have been synthesized and characterized with regard to their potential applications. Rhodamine B (RBITC) isothiocyanate was used as the fluorescent component. The core-shell structure of the particles effectively protected the dye against bleaching. HFBMA nanoparticle (NP) stability was confirmed after seven years of storage. Only slight differences were found in the polydispersity index (pdi) from 0.002 to 0.010. Particle size measurements were carried out using dynamic light scattering (DLS), nanoparticle tracking (NTA), and fluorescence correlation spectroscopy (FCS). The hydrodynamic diameter evaluated by different methods were in good agreement, respectively: 184-550 nm, 218-579 nm, and 236-508 nm. Particle and core morphology was estimated by using scanning and transmission electron microscopy (SEM and TEM). The ability to recognize particles in 3D as a reference sample in biological media has been confirmed by epifluorescence optical microscopy, confocal laser scanning microscopy, and super-resolution confocal microscopy (STED).
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Fluorocarbonos/química , Metacrilatos/química , Nanopartículas/química , Corantes Fluorescentes/química , Microscopia Confocal , Estrutura Molecular , Nanopartículas/ultraestrutura , Tamanho da Partícula , Rodaminas/química , Espectrometria de FluorescênciaRESUMO
Fish otoliths are calcium carbonate biominerals that are involved in hearing and balance sensing. An organic matrix plays a crucial role in their formation. Otolith matrix macromolecule-64 (OMM-64) is a highly acidic, calcium-binding protein (CBP) found in rainbow trout otoliths. It is a component of high-molecular-weight aggregates, which influence the size, shape and polymorph of calcium carbonate in vitro. In this study, a protocol for the efficient expression and purification of OMM-64 was developed. For the first time, the complete structural characteristics of OMM-64 were described. Various biophysical methods were combined to show that OMM-64 occurs as an intrinsically disordered monomer. Under denaturing conditions (pH, temperature) OMM-64 exhibits folding propensity. It was determined that OMM-64 binds approximately 61 calcium ions with millimolar affinity. The folding-unfolding experiments showed that calcium ions induced the collapse of OMM-64. The effect of other counter ions present in trout endolymph on OMM-64 conformational changes was studied. The significance of disordered properties of OMM-64 and the possible function of this protein is discussed.
Assuntos
Proteínas de Ligação ao Cálcio/química , Cálcio/química , Proteínas da Matriz Extracelular/química , Proteínas de Peixes/química , Proteínas Intrinsicamente Desordenadas/química , Membrana dos Otólitos/química , Animais , Sítios de Ligação , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Expressão Gênica , Concentração de Íons de Hidrogênio , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Oncorhynchus mykiss/fisiologia , Membrana dos Otólitos/metabolismo , Ligação Proteica , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Desdobramento de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TemperaturaRESUMO
It has been long known that the physical encapsulation of oleic acid-capped iron oxide nanoparticles (OA-IONPs) with the cetyltrimethylammonium (CTA+) surfactant induces the formation of spherical iron oxide nanoparticle clusters (IONPCs). However, the behavior and functional properties of IONPCs in chemical reactions have been largely neglected and are still not well-understood. Herein, we report an unconventional ligand-exchange function of IONPCs activated when dispersed in an ethyl acetate/acetate buffer system. The ligand exchange can successfully transform hydrophobic OA-IONP building blocks of IONPCs into highly hydrophilic, acetate-capped iron oxide nanoparticles (Ac-IONPs). More importantly, we demonstrate that the addition of silica precursors (tetraethyl orthosilicate and 3-aminopropyltriethoxysilane) to the acetate/oleate ligand-exchange reaction of the IONPs induces the disassembly of the IONPCs into monodispersed iron oxide-acetate-silica core-shell-shell (IONPs@acetate@SiO2) nanoparticles. Our observations evidence that the formation of IONPs@acetate@SiO2 nanoparticles is initiated by a unique micellar fusion mechanism between the Pickering-type emulsions of IONPCs and nanoemulsions of silica precursors formed under ethyl acetate buffered conditions. A dynamic rearrangement of the CTA+-oleate bilayer on the IONPC surfaces is proposed to be responsible for the templating process of the silica shells around the individual IONPs. In comparison to previously reported methods in the literature, our work provides a much more detailed experimental evidence of the silica-coating mechanism in a nanoemulsion system. Overall, ethyl acetate is proven to be a very efficient agent for an effortless preparation of monodispersed IONPs@acetate@SiO2 and hydrophilic Ac-IONPs from IONPCs.
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In this work, we describe new experimental setups for Fluorescence Correlation Spectroscopy (FCS) where a long working distance objective is used. Using these setups, FCS measurements in a broad temperature range for a small sample volume of about 50 µl can be performed. The use of specially designed cells and a dry long working distance objective was essential for avoiding temperature gradients in the sample. The performance of the new setups and a traditional FCS setup with immersion objectives is compared. The FCS data in combination with the Stokes-Einstein (SE) relation were used to obtain the values of the nanoviscosity of a fluid. We show for selected molecular van der Waals supercooled liquids that despite the fact that in these systems, a characteristic length scale can be defined, the nanoviscosity obtained from FCS is in a very good agreement with the macroscopic (rheometric) viscosity of the sample in a broad temperature range. This result corroborates the applicability of the SE relation to supercooled liquids at temperatures above 1.2 Tg. We also show that the temperature dependent size of thermoresponsive microgel particles can be determined by FCS using the designed cells and a long working distance objective in a broader size range without a need to use the correction procedure since the size correction is proportional to the square of the ratio of the hydrodynamic radius to the confocal volume size.
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In some applications the dye distribution within fluorescently labeled nanoparticles and its stability over long periods of time are important issues. In this article we study numerically and experimentally the applicability of fluorescence correlation spectroscopy (FCS) to resolve such questions. When the size of fluorescently labeled particles is comparable to or larger than the confocal volume, the effective confocal volume seen in FCS experiments is increasing. Such an effect has already been studied for uniformly labeled spherical particles. In this work we analyze the form of the FCS correlation functions (CFs) for core-labeled and shell-labeled core-shell particles. For shell-labeled particles an additional fast decay was found both in simulations and in experiments on custom-made surface-labeled particles. Universal scaling of FCS correlation times based on the squared ratio of the labeled part radius of gyration to the Gaussian radius of the beam profile was found. Recipes based on the analysis of simulated CFs, proposed for interpretation of experimental results, were successfully applied to the FCS results on suspensions of large core-labeled and surface-labeled particles.
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The shape of the Brillouin light-scattering spectrum recorded from turbid liquids is distinctly distorted compared to that from a transparent sample. The reason for this is the multiple scattering of light within the medium. The usual expression for the Brillouin spectrum does not apply to the multiple scattering situation. In this Letter, we consider a Brillouin spectrum from opaque samples composed of a distribution of spectra resulting from elementary scattering events, each occurring in single scattering vector conditions. We introduce a one-parameter test function to define the probability distribution of scattering events occurring at a given value of the scattering vector. The proposed procedure was tested on model liquids that consisted of suspensions of sub-micrometer spherical particles of different size and concentration, dispersed in different carrier liquids and studied as a function of temperature. Our analysis made it possible to account for the effect of multiple scattering and to recover the values of mechanical parameters describing the pure solvents.
RESUMO
When fluorescence correlation spectroscopy (FCS) in combination with a confocal microscope is used to determine the hydrodynamic radius a of particles comparable to or larger than the linear size σ of the confocal volume of the microscope, a correction must be used that depends on the a(2)/σ(2) ratio and the distribution of the dye within the particle. Here we present the experimental validation of the theoretically predicted approximate correction necessary for appropriate measurements of the size of uniformly fluorescently labeled spheres of radius comparable to the size of the confocal volume. We also test the approximate correction formula for different ranges of the a/σ ratio and propose a simple procedure to obtain the correct nanoparticle size from such a measurement.
Assuntos
Nanopartículas/química , Hidrodinâmica , Microscopia Confocal , Tamanho da Partícula , Espectrometria de FluorescênciaRESUMO
Fluorescence correlation spectroscopy (FCS) is frequently used to measure the self-diffusion coefficient of fluorescently labeled probes in solutions, complex media, and living cells. In a standard experiment water immersion objectives and window thickness in the range of 0.13-0.19 mm are used. We show that successful FCS measurements can be performed using samples of different refractive index placed in cells having windows of different thickness, even much thicker than nominally allowed. Different water, oil, and silicon oil immersion as well as long working distance dry objectives, equipped with the correction collar, were tested and compared. We demonstrate that the requirements for FCS experiments are less stringent than those for high resolution confocal imaging and reliable relative FCS measurements can be performed even beyond the compensation range of the objectives. All these features open new possibilities for construction of custom-made high temperature and high pressure cells for FCS.
Assuntos
Corantes Fluorescentes/química , Espectrometria de Fluorescência/instrumentação , Difusão , Refratometria , Soluções , Espectrometria de Fluorescência/métodosRESUMO
Using the Rogers-Young (RY) integral equation scheme for the static pair correlation functions combined with the liquid-phase Hansen-Verlet freezing rule, we study the generic behavior of the radial distribution function and static structure factor of monodisperse charge-stabilized suspensions with Yukawa-type repulsive particle interactions at freezing. In a related article, labeled Paper I [J. Gapinski, G. Nägele, and A. Patkowski, J. Chem. Phys. 136, 024507 (2012)], this hybrid method was used to determine two-parameter freezing lines for experimentally controllable parameters, characteristic of suspensions of charged silica spheres in dimethylformamide. A universal scaling of the RY radial distribution function maximum is shown to apply to the liquid-bcc and liquid-fcc segments of the universal freezing line. A thorough analysis is made of the behavior of characteristic distances and wavenumbers, next-neighbor particle coordination numbers, osmotic compressibility factor, and the Ravaché-Mountain-Streett minimum-maximum radial distribution function ratio.
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Coloides/química , Congelamento , Algoritmos , Modelos Químicos , Tamanho da PartículaRESUMO
Multi-responsive nanomembranes are a new class of advanced materials that can be harnessed in complex architectures for micro and nano-manipulators, artificial muscles, energy harvesting, soft robotics, and sensors. The design and fabrication of responsive membranes must meet such challenges as trade-offs between responsiveness and mechanical durability, volumetric low-cost production ensuring low environmental impact, and compatibility with standard technologies or biological systems This work demonstrates the fabrication of multi-responsive, mechanically robust poly(1,3-diaminopropane) (pDAP) nanomembranes and their application in fast photoactuators. The pDAP films are developed using a plasma-assisted polymerization technique that offers large-scale production and versatility of potential industrial relevance. The pDAP layers exhibit high elasticity with the Young's modulus of ≈7 GPa and remarkable mechanical durability across 20-80 °C temperatures. Notably, pDAP membranes reveal immediate and reversible contraction triggered by light, rising temperature, or reducing relative humidity underpinned by a reversible water sorption mechanism. These features enable the fabrication of photoactuators composed of pDAP-coated Si nanocantilevers, demonstrating ms timescale response to light, tens of µm deflections, and robust performance up to kHz frequencies. These results advance fundamental research on multi-responsive nanomembranes and hold the potential to boost versatile applications in light-to-motion conversion and sensing toward the industrial level.
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During transcription-coupled DNA repair (TCR) the detection of DNA damage and initiation of nucleotide excision repair (NER) is performed by translocating RNA polymerases (RNAP), which are arrested upon encountering bulky DNA lesions. Two opposing models of the subsequent steps of TCR in bacteria exist. In the first model, stalled RNAPs are removed from the damage site by recruitment of Mfd which dislodges RNAP by pushing it forwards before recruitment of UvrA and UvrB. In the second model, UvrD helicase backtracks RNAP from the lesion site. Recent studies have proposed that both UvrD and UvrA continuously associate with RNAP before damage occurs, which forms the primary damage sensor for NER. To test these two models of TCR in living E. coli, we applied super-resolution microscopy (PALM) combined with single particle tracking to directly measure the mobility and recruitment of Mfd, UvrD, UvrA, and UvrB to DNA during ultraviolet-induced DNA damage. The intracellular mobilities of NER proteins in the absence of DNA damage showed that most UvrA molecules could in principle be complexed with RNAP, however, this was not the case for UvrD. Upon DNA damage, Mfd recruitment to DNA was independent of the presence of UvrA, in agreement with its role upstream of this protein in the TCR pathway. In contrast, UvrD recruitment to DNA was strongly dependent on the presence of UvrA. Inhibiting transcription with rifampicin abolished Mfd DNA-recruitment following DNA damage, whereas significant UvrD, UvrA, and UvrB recruitment remained, consistent with a UvrD and UvrA performing their NER functions independently of transcribing RNAP. Together, although we find that up to â¼8 UvrD-RNAP-UvrA complexes per cell could potentially form in the absence of DNA damage, our live-cell data is not consistent with this complex being the primary DNA damage sensor for NER.
Assuntos
Proteínas de Escherichia coli , Escherichia coli , Escherichia coli/genética , Escherichia coli/metabolismo , Fatores de Transcrição/metabolismo , Imagem Individual de Molécula , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Bactérias/metabolismo , Reparo do DNA , Dano ao DNA , RNA Polimerases Dirigidas por DNA/metabolismo , DNA/metabolismo , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , DNA Helicases/metabolismoRESUMO
HYPOTHESIS: Polymer particles self-assembled into colloidal crystals have exciting applications in photonics, phononics, templates for nanolithography, and coatings. Cold soldering utilizing polymer plasticization by supercritical fluids enables a novel, low-cost, low-effort, chemical-free means for uniform mechanical strengthening of fragile polymer colloidal crystals at moderate temperatures. Here, we aim to elucidate the role of particle size and gas-specific response for the most efficient soldering, exploring the full potential of this method. EXPERIMENTS: We investigate the elastic properties of polystyrene colloidal crystals made of nanoparticles with different diameters (143 to 830 nm) upon treatment with supercritical Ar and He at room temperature. By employing Brillouin light scattering, we quantify the effect of nanoparticle size on the strengthening of interparticle contacts, evaluating the permanent change in the effective elastic modulus upon cold soldering. FINDINGS: The relative change in the effective elastic modulus reveals nonmonotonic dependence on the particle size with the most efficient soldering for mid-sized nanoparticles (about 610 nm diameter). We attribute this behavior to the crucial role of intrinsic fabrication impurities, which reduces the nanoparticles' free surface exposed to plasticization by supercritical fluids. Supercritical Ar, a good solvent for polystyrene, enabled effective soldering of nanoparticles, whereas high-pressure He treatment is entirely reversible.
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Nanopartículas , Poliestirenos , Poliestirenos/química , Coloides/química , Nanopartículas/química , Solventes/química , TemperaturaRESUMO
Aggregation of the polydopamine (PDA) molecular building blocks at the air/water interface leads to obtaining large surface nanometric-thin films. This mechanism follows two possible pathways, namely, covalent or non-covalent self-assembly, which result in a different degree of structure order and, consequently, different structural properties. Control of this mechanism could be vital for applications that require true self-support PDA free-standing films, for example, electrochemical sensing or membrane technology. Here, we are considering the impact of boric acid (BA) and Cu2+ ions on the mentioned mechanism exclusively for the free-standing films from the air/water interface. We have employed and refined our own spectroscopic reflectometry method to achieve an exceptionally high real-time control over the thickness growth. It turned out that BA and Cu2+ ions significantly impact the film growth process. Reduction of the nanoparticles size and their number was examined via UV-vis spectroscopy and transmission electron microscopy, showing a colossal reduction in the mean diameter of nanoparticles in the case of BA and a moderate reduction in the case of Cu2+. This modification is leading to significant enhancement of the process efficiency through moderation of the topological properties of the films, as revealed by atomic force microscopy. Next, applying infrared, Raman, and X-ray photoelectron spectroscopy, we presented small amounts of metal (B or Cu) in the final structure of PDA and simultaneously their vital role in the oxidation mechanism and cross-linking through covalent or non-covalent bonds. Therefore, we revealed the possibility of synthesizing films via the expected self-assembly mechanism which has hitherto been out of control. Moreover, modification of mechanical properties toward exceptionally elastic films through the BA-assisted synthesis pathway was shown by achieving Young's modulus value up to 24.1 ± 5.6 and 18.3 ± 6.4 GPa, using nanoindentation and Brillouin light scattering, respectively.
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Using the Rogers-Young (RY) integral equation scheme for the static structure factor combined with the one-phase Hansen-Verlet (HV) freezing rule, we study the equilibrium structure and two-parameter freezing lines of colloidal particles with Yukawa-type pair interactions representing charge-stabilized silica spheres suspended in dimethylformamide (DMF). Results are presented for a vast range of concentrations, salinities and effective charges covering particles with masked excluded-volume interactions. The freezing lines were obtained for the low-charge and high-charge solutions of the static structure factor, for various two-parameter sets of experimentally accessible system parameters. All RY-HV based freezing lines can be mapped on a universal fluid-solid coexistence line in good agreement with computer simulation predictions. The RY-HV calculations extend the freezing lines obtained in earlier simulations to a broader parameter range. The experimentally observed fluid-bcc-fluid reentrant transition of charged silica spheres in DMF can be explained using the freezing lines obtained in this work.
Assuntos
Algoritmos , Dimetilformamida/química , Congelamento , Dióxido de Silício/química , Coloides/química , Simulação por ComputadorRESUMO
We present a scaling formula for size-dependent viscosity coefficients for proteins, polymers, and fluorescent dyes diffusing in complex liquids. The formula was used to analyze the mobilities of probes of different sizes in HeLa and Swiss 3T3 mammalian cells. This analysis unveils in the cytoplasm two length scales: (i) the correlation length ξ (approximately 5 nm in HeLa and 7 nm in Swiss 3T3 cells) and (ii) the limiting length scale that marks the crossover between nano- and macroscale viscosity (approximately 86 nm in HeLa and 30 nm in Swiss 3T3 cells). During motion, probes smaller than ξ experienced matrix viscosity: η(matrix) ≈ 2.0 mPa·s for HeLa and 0.88 mPa·s for Swiss 3T3 cells. Probes much larger than the limiting length scale experienced macroscopic viscosity, η(macro) ≈ 4.4 × 10(-2) and 2.4 × 10(-2) Pa·s for HeLa and Swiss 3T3 cells, respectively. Our results are persistent for the lengths scales from 0.14 nm to a few hundred nanometers.
Assuntos
Citoplasma/metabolismo , Nanotecnologia/métodos , Células 3T3 , Animais , Linhagem Celular , Difusão , Células HeLa , Humanos , Camundongos , Micelas , Peso Molecular , Nanopartículas/química , Polímeros/química , Poliestirenos/química , Pressão , ViscosidadeRESUMO
Hydrophobicity is one of the most critical factors governing the adsorption of molecules and objects, such as virions, on surfaces. Even moderate change of wetting angle of plastic surfaces causes a drastic decrease ranging from 2 to 5 logs of the viruses (e.g., T4 phage) in the suspension due to adsorption on polymer vials' walls. The effect varies immensely in seemingly identical containers but purchased from different vendors. Comparison of glass, polyethylene, polypropylene, and polystyrene containers revealed a threshold in the wetting angle of around 95°: virions adsorb on the surface of more hydrophobic containers, while in more hydrophilic vials, phage suspensions are stable. The polypropylene surface of the Eppendorf-type and Falcon-type can accommodate from around 108 PFU/ml to around 1010 PFU/ml from the suspension. The adsorption onto the container's wall might result in complete scavenging of virions from the bulk. We developed two methods to overcome this issue. The addition of surfactant Tween20 and/or plasma treatment provides a remedy by modulating surface wettability and inhibiting virions' adsorption. Plastic containers are essential consumables in the daily use of many bio-laboratories. Thus, this is important not only for phage-related research (e.g., the use of phage therapies as an alternative for antibiotics) but also for data comparison and reproducibility in the field of biochemistry and virology.
Assuntos
Bacteriófagos/metabolismo , Polipropilenos/química , Adsorção , Bacteriófago T4 , Vidro/química , Interações Hidrofóbicas e Hidrofílicas , Microscopia de Força Atômica , Microscopia Eletrônica de Varredura , Espectroscopia Fotoeletrônica , Plásticos , Polietileno/química , Polímeros/química , Polissorbatos/química , Poliestirenos/química , Reprodutibilidade dos Testes , Propriedades de Superfície , Tensoativos , Temperatura , Vírion , MolhabilidadeRESUMO
In this work, we show synthesis that leads to thermoreponsive poly-N-isopropyl acrylamide (pNIPAM) nanogels with sizes below 100 nm, irrespectively of the surfactant to crosslinker ratio. We also show that in many environments the temperature induced pNIPAM collapse at Lower Critical Solution Temperature (LCST) of 32.5 °C is accompanied by gel nanoparticles' aggregation. Thus, the proper information on the nanoparticle (NP) structure and deswelling can be obtained only if the routinely measured hydrodynamic radius is supplemented by information on the molecular weight, which can be obtained from the intensity of scattered light. We measured the dynamics and reversibility of the deswelling and subsequent aggregation processes. Furthermore, we show that the highly concentrated pNIPAM gel NPs reversibly form bulk hydrogel networks of varied interconnected porous structure. We show, that in case of drying pNIPAM gel NPs above the LCST, it is possible to obtain films with 20-fold increase in storage modulus (G') compared to hydrogel networks measured at room temperature. They exhibit temperature hysteresis behavior around LCST of 32.5 °C similar to pNIPAM films. Finally, we show that these hydrogel films, lead to extended proliferation of cells across three different types: fibroblast, endothelial and cancer cells. Additionally, none of the films exhibited any cytotoxic effects. Our study brings new insights into physicochemical characterization of pNIPAM gel NPs and networks behavior in realistic conditions of in vitro measurements, especially by means of dynamic light scattering as well as final unique properties of both gel NPs and formed porous films for possible tissue engineering applications.