RESUMO
We herein address the problem of polymorph selection by introducing a general and straightforward concept based on their ordering. We demonstrated the concept by the ordered patterning of four compounds capable of forming different polymorphs when deposited on technologically relevant surfaces. Our approach exploits the fact that, when the growth of a crystalline material is confined within sufficiently small cavities, only one of the possible polymorphs is generated. We verify our method by utilizing several model compounds to fabricate micrometric "logic patterns" in which each of the printed pixels is easily identifiable as comprising only one polymorph and can be individually accessed for further operations.
RESUMO
MOTIVATION: Single-molecule force spectroscopy (SMFS) experiments pose the challenge of analysing protein unfolding data (traces) coming from preparations with heterogeneous composition (e.g. where different proteins are present in the sample). An automatic procedure able to distinguish the unfolding patterns of the proteins is needed. Here, we introduce a data analysis pipeline able to recognize in such datasets traces with recurrent patterns (clusters). RESULTS: We illustrate the performance of our method on two prototypical datasets: â¼50 000 traces from a sample containing tandem GB1 and â¼400 000 traces from a native rod membrane. Despite a daunting signal-to-noise ratio in the data, we are able to identify several unfolding clusters. This work demonstrates how an automatic pattern classification can extract relevant information from SMFS traces from heterogeneous samples without prior knowledge of the sample composition. AVAILABILITY AND IMPLEMENTATION: https://github.com/ninailieva/SMFS_clustering. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Proteínas , Imagem Individual de Molécula , Desdobramento de Proteína , Razão Sinal-Ruído , Software , Análise EspectralRESUMO
The mechanical properties of biogenic membranous compartments are thought to be relevant in numerous biological processes; however, their quantitative measurement remains challenging for most of the already available force spectroscopy (FS)-based techniques. In particular, the debate on the mechanics of lipid nanovesicles and on the interpretation of their mechanical response to an applied force is still open. This is mostly due to the current lack of a unified model being able to describe the mechanical response of both gel and fluid phase lipid vesicles and to disentangle the contributions of membrane rigidity and luminal pressure. In this framework, we herein propose a simple model in which the interplay of membrane rigidity and luminal pressure to the overall vesicle stiffness is described as a series of springs; this approach allows estimating these two contributions for both gel and fluid phase liposomes. Atomic force microscopy-based FS, performed on both vesicles and supported lipid bilayers, is exploited for obtaining all the parameters involved in the model. Moreover, the use of coarse-grained full-scale molecular dynamics simulations allowed for better understanding of the differences in the mechanical responses of gel and fluid phase bilayers and supported the experimental findings. The results suggest that the pressure contribution is similar among all the probed vesicle types; however, it plays a dominant role in the mechanical response of lipid nanovesicles presenting a fluid phase membrane, while its contribution becomes comparable to the one of membrane rigidity in nanovesicles with a gel phase lipid membrane. The results presented herein offer a simple way to quantify two of the most important parameters in vesicle nanomechanics (membrane rigidity and internal pressurization), and as such represent a first step toward a currently unavailable, unified model for the mechanical response of gel and fluid phase lipid nanovesicles.
Assuntos
Fenômenos Biológicos , Bicamadas Lipídicas , Lipossomos , Fenômenos Mecânicos , Microscopia de Força AtômicaRESUMO
The mechanical properties of extracellular vesicles (EVs) are known to influence their biological function, in terms of, e.g., cellular adhesion, endo/exocytosis, cellular uptake, and mechanosensing. EVs have a characteristic nanomechanical response which can be probed via force spectroscopy (FS) and exploited to single them out from nonvesicular contaminants or to discriminate between subtypes. However, measuring the nanomechanical characteristics of individual EVs via FS is a labor-intensive and time-consuming task, usually limiting this approach to specialists. Herein, we describe a simple atomic force microscopy based experimental procedure for the simultaneous nanomechanical and morphological analysis of several hundred individual nanosized EVs within the hour time scale, using basic AFM equipment and skills and only needing freely available software for data analysis. This procedure yields a "nanomechanical snapshot" of an EV sample which can be used to discriminate between subpopulations of vesicular and nonvesicular objects in the same sample and between populations of vesicles with similar sizes but different mechanical characteristics. We demonstrate the applicability of the proposed approach to EVs obtained from three very different sources (human colorectal carcinoma cell culture, raw bovine milk, and Ascaris suum nematode excretions), recovering size and stiffness distributions of individual vesicles in a sample. EV stiffness values measured with our high-throughput method are in very good quantitative accord with values obtained by FS techniques which measure EVs one at a time. We show how our procedure can detect EV samples contamination by nonvesicular aggregates and how it can quickly attest the presence of EVs even in samples for which no established assays and/or commercial kits are available (e.g., Ascaris EVs), thus making it a valuable tool for the rapid assessment of EV samples during the development of isolation/enrichment protocols by EV researchers. As a side observation, we show that all measured EVs have a strikingly similar stiffness, further reinforcing the hypothesis that their mechanical characteristics could have a functional role.
Assuntos
Vesículas Extracelulares/química , Ensaios de Triagem em Larga Escala , Microscopia de Força Atômica , Nanotecnologia , Animais , Ascaris suum/química , Bovinos , Células HCT116 , Humanos , Lipossomos/química , Leite/químicaRESUMO
Inorganic nanoparticles (NPs) represent promising examples of engineered nanomaterials, providing interesting biomedical solutions in several fields, like therapeutics and diagnostics. Despite the extensive number of investigations motivated by their remarkable potential for nanomedicinal applications, the interactions of NPs with biological interfaces are still poorly understood. The effect of NPs on living organisms is mediated by biological barriers, such as the cell plasma membrane, whose lateral heterogeneity is thought to play a prominent role in NPs adsorption and uptake pathways. In particular, biological membranes feature the presence of rafts, that is segregated lipid micro and/or nanodomains in the so-called liquid ordered phase (Lo ), immiscible with the surrounding liquid disordered phase (Ld ). Rafts are involved in various biological functions and act as sites for the selective adsorption of materials on the membrane. Indeed, the thickness mismatch present along their boundaries generates energetically favourable conditions for the adsorption of NPs. Despite its clear implications in NPs internalisation processes and cytotoxicity, a direct proof of the selective adsorption of NPs along the rafts' boundaries is still missing to date. Here we use multicomponent supported lipid bilayers (SLBs) as reliable synthetic models, reproducing the nanometric lateral heterogeneity of cell membranes. After being characterised by atomic force microscopy (AFM) and neutron reflectivity (NR), multidomain SLBs are challenged by prototypical inorganic nanoparticles, that is citrated gold nanoparticles (AuNPs), under simplified and highly controlled conditions. By exploiting AFM, we demonstrate that AuNPs preferentially target lipid phase boundaries as adsorption sites. The herein reported study consolidates and extends the fundamental knowledge on NPs-membrane interactions, which constitute a key aspect to consider when designing NPs-related biomedical applications. LAY DESCRIPTION: Inorganic nanoparticles (NPs) represent promising examples of engineered nanomaterials, offering interesting biomedical solutions in multiple fields like therapeutics and diagnostics. Despite being extensively investigated due to their remarkable potential for nanomedicinal applications, the interaction of NPs with biological systems is in several cases still poorly understood. The interaction of NPs with living organisms is mediated by biological barriers, such as the cell plasma membrane. Supported lipid bilayers (SLBs) represent suitable synthetic membrane models for studying the physicochemical properties of natural interfaces and their interaction with inorganic nanomaterials under simplified and controlled conditions. Recently, multicomponent SLBs were developed in order to mimic the lateral heterogeneity of most biological membranes. In particular, biological membranes feature the presence of rafts, that is segregated lipid micro and/or nanodomains, enriched in cholesterol, sphingomyelin, saturated glycerophospholipids and glycosphingolipids: these lipids segregate in the so-called liquid-ordered phase (Lo ), characterised by a high molecular packing degree, which promotes the phase separation from the surrounding liquid-crystalline (disordered, Ld ) phase, where the intermolecular mobility is increased. Rafts are thought to participate in the formation and targeting of nano-sized biogenic lipid vesicles and are also actively involved in multiple membrane processes. Indeed, Lo -Ld phase boundaries represent high energy areas, providing active sites for the preferential adsorption of external material. The selective adsorption of NPs along the phase boundaries of rafted membranes has been theorised and indirectly probed by different research groups; however, a direct proof of this phenomenon is still missing to date. We herein exploit atomic force microscopy (AFM) to directly visualise the preferential adsorption of gold nanoparticles (AuNPs) along the phase boundaries of multicomponent SLBs (previously characterised by neutron reflectivity), obtained from synthetic vesicles containing both an Ld and an Lo phase. The quantitative localisation and morphometry of AuNPs adsorbed on the SLB reveal important information on their interaction with the lipid matrix and directly prove the already theorised differential NPs-lipid interaction at the phase boundaries. The presented results could help the development of future NP-based applications, involving NPs adsorption on membranes with nanoscale phase segregations.
Assuntos
Ouro/química , Bicamadas Lipídicas/química , Microdomínios da Membrana/química , Nanopartículas Metálicas/química , Microscopia de Força Atômica/métodos , Membrana Celular/metabolismoRESUMO
We investigate the solvatochromic effect of a Fe-based spin-crossover (SCO) compound via ambient pressure soft X-ray absorption spectroscopy (AP-XAS) and atomic force microscopy (AFM). AP-XAS provides the direct evidence of the spin configuration for the Fe(II) 3d states of the SCO material upon in situ exposure to specific gas or vapor mixtures; concurrent changes in nanoscale topography and mechanical characteristics are revealed via AFM imaging and AFM-based force spectroscopy, respectively. We find that exposing the SCO material to gaseous helium promotes an effective decrease of the transition temperature of its surface layers, while the exposure to methanol vapor causes opposite surfacial and bulk solvatochromic effects. Surfacial solvatochromism is accompanied by a dramatic reduction of the surface layers stiffness. We propose a rationalization of the observed effects based on interfacial dehydration and solvation phenomena.
RESUMO
The analysis of the α-synuclein (aS) aggregation process, which is involved in Parkinson's disease etiopathogenesis, and of the structural feature of the resulting amyloid fibrils may shed light on the relationship between the structure of aS aggregates and their toxicity. This may be considered a paradigm of the ground work needed to tackle the molecular basis of all the protein-aggregation-related diseases. With this aim, we used chemical and physical dissociation methods to explore the structural organization of wild-type aS fibrils. High pressure (in the kbar range) and alkaline pH were used to disassemble fibrils to collect information on the hierarchic pathway by which distinct ß-sheets sequentially unfold using the unique possibility offered by high-pressure Fourier transform infrared spectroscopy. The results point toward the formation of kinetic traps in the energy landscape of aS fibril disassembly and the presence of transient partially folded species during the process. Since we found that the dissociation of wild-type aS fibrils by high pressure is reversible upon pressure release, the disassembled molecules likely retain structural information that favors fibril reformation. To deconstruct the role of the different regions of aS sequence in this process, we measured the high-pressure dissociation of amyloids formed by covalent chimeric dimers of aS (syn-syn) and by the aS deletion mutant that lacks the C-terminus, i.e., aS (1-99). The results allowed us to single out the role of dimerization and that of the C-terminus in the complete maturation of fibrillar aS.
Assuntos
Amiloide/metabolismo , alfa-Sinucleína/metabolismo , Amiloide/química , Dicroísmo Circular , Escherichia coli , Concentração de Íons de Hidrogênio , Microscopia de Força Atômica , Microscopia Eletrônica de Transmissão , Modelos Moleculares , Mutação , Pressão , Conformação Proteica em Folha beta , Domínios Proteicos , Dobramento de Proteína , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Espectroscopia de Infravermelho com Transformada de Fourier , Análise Espectral Raman , alfa-Sinucleína/químicaRESUMO
Familial and idiopathic Parkinson's disease (PD) is associated with the abnormal neuronal accumulation of α-synuclein (aS) leading to ß-sheet-rich aggregates called Lewy Bodies (LBs). Moreover, single point mutation in aS gene and gene multiplication lead to autosomal dominant forms of PD. A connection between PD and the 14-3-3 chaperone-like proteins was recently proposed, based on the fact that some of the 14-3-3 isoforms can interact with genetic PD-associated proteins such as parkin, LRRK2 and aS and were found as components of LBs in human PD. In particular, a direct interaction between 14-3-3η and aS was reported when probed by co-immunoprecipitation from cell models, from parkinsonian brains and by surface plasmon resonance in vitro. However, the mechanisms through which 14-3-3η and aS interact in PD brains remain unclear. Herein, we show that while 14-3-3η is unable to bind monomeric aS, it interacts with aS oligomers which occur during the early stages of aS aggregation. This interaction diverts the aggregation process even when 14-3-3η is present in sub-stoichiometric amounts relative to aS. When aS level is overwhelmingly higher than that of 14-3-3η, the fibrillation process becomes a sequestration mechanism for 14-3-3η, undermining all processes governed by this protein. Using a panel of complementary techniques, we single out the stage of aggregation at which the aS/14-3-3η interaction occurs, characterize the products of the resulting processes, and show how the processes elucidated in vitro are relevant in cell models. Our findings constitute a first step in elucidating the molecular mechanism of aS/14-3-3η interaction and in understanding the critical aggregation step at which 14-3-3η has the potential to rescue aS-induced cellular toxicity.
Assuntos
Proteínas 14-3-3/metabolismo , Amiloidose/metabolismo , Agregação Patológica de Proteínas , Transdução de Sinais , alfa-Sinucleína/metabolismo , Proteínas 14-3-3/química , Proteínas 14-3-3/genética , Humanos , Cinética , Ligação Proteica , Isoformas de Proteínas , alfa-Sinucleína/química , alfa-Sinucleína/genéticaRESUMO
This article reports on the synthesis of an innovative smart polymer, P5-QPDMAEMA, opportunely developed with the aim of combining the responsiveness of PDMAEMA polymer and the host-guest properties of covalently linked pillar[5]arenes. Thanks to a traditional Non-Induced Phase Separation (NIPS) process performed at various coagulation pH, the blending of P5-QPDMAEMA with polyethersulfone gave rise to the formation of functional beads for the removal of organic dyes in water. Adsorption tests are carried out on all the produced blend-based beads by employing two representative dyes, the cationic methylene blue (MB), and the anionic methyl orange (MO). In particular, the P5-QPDMAEMA based beads, prepared at acidic pH, featured the best MO removal rate (i. e., 91.3 % after 150â minutes starting from a 20â mg â L-1 solution) and a high selectivity towards the removal of the selected anionic dye. Based on the adsorption kinetics and isotherm calculations, the pseudo-first order and Freundlich models were shown to be the most suitable to describe the MO adsorption behavior, achieving a maximum adsorption capacity of 21.54â mg â g-1. Furthermore, zwitterionic beads are obtained by a post-functionalization of the PDMAEMA and the P5-QPDMAEMA based beads, to test their removal capability towards both anionic and cationic dyes, as shown.
RESUMO
Since the SARS-CoV-2 virus started spreading worldwide, evidence pointed toward an impact of the infection on the nervous system. COVID-19 patients present neurological manifestations and have an increased risk of developing brain-related symptoms in the long term. In fact, evidence in support of the neuroinvasive potential of SARS-CoV-2 has emerged. Considering that viral parkisonism was observed as a consequence of encephalopathies caused by viral infections, it has been already suggested that COVID-19 could affect the dopaminergic neurons and contribute to neurodegeneration in Parkinson's disease (PD), by promoting the formation of amyloid fibrils constituted by the PD-related protein α-synuclein. Here, we observe not only that SARS-CoV-2 viral spike protein and nucleocapsid protein can alone promote α-synuclein aggregation but also that the spike protein organization in a corona shape on the viral envelope may be crucial in triggering fast amyloid fibrils formation, thus possibly contributing to PD pathogenesis.
Assuntos
COVID-19 , Doença de Parkinson , Humanos , alfa-Sinucleína/metabolismo , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Doença de Parkinson/metabolismoRESUMO
Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly.
Assuntos
Exossomos , Vesículas Extracelulares , Vesículas Extracelulares/metabolismo , Exossomos/metabolismo , Transporte Biológico , Biomarcadores/metabolismo , FenótipoRESUMO
A stable and improved control of the wettability of textiles was obtained by using a coating of diamond like carbon (DLC) films on cotton by PECVD. By controlling different plasma pretreatments of argon, oxygen, and hydrogen on the cotton fibers' surface, we have shown that the pretreatments had a significant impact on wettability behavior resulting from an induced nanoscale roughness combined with an incorporation of selected functional groups. Upon subsequent deposition of diamond like carbon (DLC) films, the cotton fibers yield to a highly controlled chemical stability and hydrophobic state and could be used for self-cleaning applications. By controlling the nature of the plasma pretreatment we have shown that the oxygen plasma pretreatment was more effective than the argon and hydrogen for the superhydrophilic/ultra hydrophobic properties. The chemical and morphological changes of the cotton fibers under different treatments were characterized using X-ray photoelectron and Raman spectroscopy, AFM, and water contact angle measurements. The mechanism underlying the water-repellent properties of the cotton fibers provides a new and innovative pathway into the development of a range of advanced self-cleaning textiles.
Assuntos
Carbono/química , Fibra de Algodão , Argônio/química , Hidrogênio/química , Interações Hidrofóbicas e Hidrofílicas , Oxigênio/química , Tamanho da Partícula , Propriedades de Superfície , MolhabilidadeRESUMO
Although promising for biomedicine, the clinical translation of inorganic nanoparticles (NPs) is limited by low biocompatibility and stability in biological fluids. A common strategy to circumvent this drawback consists in disguising the active inorganic core with a lipid bilayer coating, reminiscent of the structure of the cell membrane to redefine the chemical and biological identity of NPs. While recent reports introduced membrane-coating procedures for NPs, a robust and accessible method to quantify the integrity of the bilayer coverage is not yet available. To fill this gap, we prepared SiO2 nanoparticles (SiO2NPs) with different membrane coverage degrees and monitored their interaction with AuNPs by combining microscopic, scattering, and optical techniques. The membrane-coating on SiO2NPs induces spontaneous clustering of AuNPs, whose extent depends on the coating integrity. Remarkably, we discovered a linear correlation between the membrane coverage and a spectral descriptor for the AuNPs' plasmonic resonance, spanning a wide range of coating yields. These results provide a fast and cost-effective assay to monitor the compatibilization of NPs with biological environments, essential for bench tests and scale-up. In addition, we introduce a robust and scalable method to prepare SiO2NPs/AuNPs hybrids through spontaneous self-assembly, with a high-fidelity structural control mediated by a lipid bilayer.
Assuntos
Nanopartículas Metálicas , Nanopartículas , Bicamadas Lipídicas/química , Nanopartículas Metálicas/química , Ouro/química , Dióxido de Silício/química , Biomimética , Nanopartículas/químicaRESUMO
The widely overlapping physicochemical properties of lipoproteins (LPs) and extracellular vesicles (EVs) represents one of the main obstacles for the isolation and characterization of these pervasive biogenic lipid nanoparticles. We herein present the application of an atomic force microscopy (AFM)-based quantitative morphometry assay to the rapid nanomechanical screening of mixed LPs and EVs samples. The method can determine the diameter and the mechanical stiffness of hundreds of individual nanometric objects within few hours. The obtained diameters are in quantitative accord with those measured via cryo-electron microscopy (cryo-EM); the assignment of specific nanomechanical readout to each object enables the simultaneous discrimination of co-isolated EVs and LPs even if they have overlapping size distributions. EVs and all classes of LPs are shown to be characterised by specific combinations of diameter and stiffness, thus making it possible to estimate their relative abundance in EV/LP mixed samples in terms of stoichiometric ratio, surface area and volume. As a side finding, we show how the mechanical behaviour of specific LP classes is correlated to distinctive structural features revealed by cryo-EM. The described approach is label-free, single-step and relatively quick to perform. Importantly, it can be used to analyse samples which prove very challenging to assess with several established techniques due to ensemble-averaging, low sensibility to small particles, or both, thus providing a very useful tool for quickly assessing the purity of EV/LP isolates including plasma- and serum-derived preparations.
Assuntos
Vesículas Extracelulares , Microscopia Crioeletrônica , Vesículas Extracelulares/química , Microscopia de Força Atômica/métodos , Lipopolissacarídeos , Lipoproteínas/análiseRESUMO
Extracellular vesicles (EVs) in blood plasma are recognized as potential biomarkers for disease. Although blood plasma is easily obtainable, analysis of EVs at the single particle level is still challenging due to the biological complexity of this body fluid. Besides EVs, plasma contains different types of lipoproteins particles (LPPs), that outnumber EVs by orders of magnitude and which partially overlap in biophysical properties such as size, density and molecular makeup. Consequently, during EV isolation LPPs are often co-isolated. Furthermore, physical EV-LPP complexes have been observed in purified EV preparations. Since co-isolation or association of LPPs can impact EV-based analysis and biomarker profiling, we investigated the presence and formation of EV-LPP complexes in biological samples by using label-free atomic force microscopy, cryo-electron tomography and synchronous Rayleigh and Raman scattering analysis of optically trapped particles and fluorescence-based high sensitivity single particle flow cytometry. Furthermore, we evaluated the impact on flow cytometric analysis in the presence of LPPs using in vitro spike-in experiments of purified tumour cell line-derived EVs in different classes of purified human LPPs. Based on orthogonal single-particle analysis techniques we demonstrate that EV-LPP complexes can form under physiological conditions. Furthermore, we show that in fluorescence-based flow cytometric EV analysis staining of LPPs, as well as EV-LPP associations, can influence quantitative and qualitative EV analysis. Lastly, we demonstrate that the colloidal matrix of the biofluid in which EVs reside impacts their buoyant density, size and/or refractive index (RI), which may have consequences for down-stream EV analysis and EV biomarker profiling.
Assuntos
Vesículas Extracelulares , Humanos , Vesículas Extracelulares/fisiologia , Imagem Individual de Molécula , Biomarcadores , Linhagem Celular Tumoral , Lipoproteínas LDLRESUMO
We show via single-molecule mechanical unfolding experiments that the osmolyte glycerol stabilizes the native state of the human cardiac I27 titin module against unfolding without shifting its unfolding transition state on the mechanical reaction coordinate. Taken together with similar findings on the immunoglobulin-binding domain of streptococcal protein G (GB1), these experimental results suggest that osmolytes act on proteins through a common mechanism that does not entail a shift of their unfolding transition state. We investigate the above common mechanism via an Ising-like model for protein mechanical unfolding that adds worm-like-chain behavior to a recent generalization of the Wako-Saitô-Muñoz-Eaton model with support for group-transfer free energies. The thermodynamics of the model are exactly solvable, while protein kinetics under mechanical tension can be simulated via Monte Carlo algorithms. Notably, our force-clamp and velocity-clamp simulations exhibit no shift in the position of the unfolding transition state of GB1 and I27 under the effect of various osmolytes. The excellent agreement between experiment and simulation strongly suggests that osmolytes do not assume a structural role at the mechanical unfolding transition state of proteins, acting instead by adjusting the solvent quality for the protein chain analyte.
Assuntos
Fenômenos Mecânicos , Modelos Moleculares , Osmose , Desdobramento de Proteína , Fenômenos Biomecânicos , Glicerol/química , Cinética , Conformação Proteica , TermodinâmicaRESUMO
MOTIVATION: Single-molecule force spectroscopy has facilitated the experimental investigation of biomolecular force-coupled kinetics, from which the kinetics at zero force can be extrapolated via explicit theoretical models. The atomic force microscope (AFM) in particular is routinely used to study protein unfolding kinetics, but only rarely protein folding kinetics. The discrepancy arises because mechanical protein refolding studies are more technically challenging. RESULTS: We developed software that can drive and analyse mechanical refolding experiments when used with the commercial AFM setup 'Picoforce AFM', Bruker (previously Digital Instruments). We expect the software to be easily adaptable to other AFM setups. We also developed an improved method for the statistical characterization of protein folding kinetics, and implemented it into an AFM-independent software module. AVAILABILITY: Software and documentation are available at http://code.google.com/p/refolding under Apache License 2.0.
Assuntos
Biologia Computacional/métodos , Redobramento de Proteína , Software , Cinética , Desnaturação ProteicaRESUMO
HYPOTHESIS: The broad detection properties of alizarin, not only concerning pH variations but also temperature, glucose and health-like relevant cations alterations, make it a molecule of great scientific interest, particularly for developing multifunctional wearable sensors. EXPERIMENT: Herein, the alizarin red S dyestuff is bonded with trimethoxy-[3-(oxiran-2-ylmethoxy)propyl]silane, as a sol-gel precursor, to functionalize cotton fabrics. The chemical and structural properties of both plain and silane-functionalized dyestuffs are investigated in solution and solid-state by several chemical-physical characterization techniques. FINDINGS: The hybrid dyestuff characterization reveals the epoxy ring-opening of the silica precursor, leading to covalent linkages to the sulfonic group of alizarin, which retains its structure during the sol-gel reaction. The silane-functionalized halochromic dyestuff shows similar halochromic behaviour as its pristine solution in the investigated pH range, thus demonstrating a color shift from yellow to red due to the protonation/deprotonation reversible mechanism of the chromophore. The reversibility and repeatability of pH-sensing properties of treated cotton fabrics are confirmed by diffuse reflectance and CIELAB color space characterizations. Cotton fabric functionalized with alizarin-containing sol-gel coating shows excellent durability of halochromic properties, thus emerging as a versatile platform for stimuli-responsive materials.
Assuntos
Silanos , Dispositivos Eletrônicos Vestíveis , Antraquinonas , Silanos/química , TêxteisRESUMO
In the field of stimuli-responsive materials, introducing a pH-sensitive dyestuff onto textile fabrics is a promising approach for the development of wearable sensors. In this paper, the alizarin red S dyestuff bonded with a sol-gel precursor, namely trimethoxy-[3-(oxiran-2-ylmethoxy)propyl]silane, was used to functionalize polyethylene terephthalate fabrics, a semi-crystalline thermoplastic polyester largely used in the healthcare sector mainly due to its advantages, including mechanical strength, biocompatibility and resistance against abrasion and chemicals. The obtained hybrid halochromic silane-based coating on polyester fabrics was investigated with several chemical characterization techniques. Fourier transform infrared spectroscopy and X-ray Photoelectron Spectroscopy confirmed the immobilization of the dyestuff-based silane matrix onto polyethylene terephthalate samples through self-condensation of hydrolyzed silanols under the curing process. The reversibility and repeatability of pH-sensing properties of treated polyester fabrics in the pH range 2.0-8.0 were confirmed with diffuse reflectance and CIELAB color space characterizations. Polyester fabric functionalized with halochromic silane-based coating shows the durability of halochromic properties conversely to fabric treated with plain alizarin red S, thus highlighting the potentiality of the sol-gel approach in developing durable halochromic coating on synthetic substrates. The developed wearable pH-meter device could find applications as a non-invasive pH sensor for wellness and healthcare fields.
RESUMO
The mechanical response of lipid membranes to nanoscale deformations is of fundamental importance for understanding how these interfaces behave in multiple biological processes; in particular, the nanoscale mechanics of non-lamellar membranes represents a largely unexplored research field. Among these mesophases, inverse bicontinuous cubic phase QII membranes have been found to spontaneously occur in stressed or virally infected cells and to play a role in fundamental processes, such as cell fusion and food digestion. We herein report on the fabrication of thin ( Ì´150 nm) supported QII cubic phase lipid films (SQIIFs) and on their characterization via multiple techniques including Small Angle X-Ray Scattering (SAXS), Ellipsometry and Atomic Force Microscopy (AFM). Moreover, we present the first nanomechanical characterization of a cubic phase lipid membrane, through AFM-based Force Spectroscopy (AFM-FS). Our analysis reveals that the mechanical response of these architectures is strictly related to their topology and structure. The observed properties are strikingly similar to those of macroscopic 3D printed cubic structures when subjected to compression tests in material science; suggesting that this behaviour depends on the 3D organisation, rather than on the length-scale of the architecture. We also show for the first time that AFM-FS can be used for characterizing the structure of non-lamellar mesophases, obtaining lattice parameters in agreement with SAXS data. In contrast to classical rheological studies, which can only probe bulk cubic phase solutions, our AFM-FS analysis allows probing the response of cubic membranes to deformations occurring at length and force scales similar to those found in biological interactions.