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Following a new approach, we prepared a nanoink with two separate photothermally responsive absorption bands. One is the localized surface plasmon resonance (LSPR) absorption of gold nanoparticles (AuNP, d=17â nm), the second is the absorption band of two cyanine (Cy) dyes, Cy7-C6 or Cy7-C11, grafted to the AuNP surface through thiolated bridges of different lengths: the close proximity to the Au surface induces full quenching of the Cy fluorescence, resulting in thermal relaxation on irradiation. Attempts to full coat AuNP with the lipophilic Cy7-C6 and Cy7-C11 lead to precipitation from aqueous solutions. We thus prepared AuNP with partial pegylation (30, 50, or 70 %), using a long chain thiol-terminated PEG bearing a -COOH function. Addition until saturation of either Cy7-C6 or Cy7-C11 to the partially pegylated AuNP gave the AuNP@Cy/PEGX% hybrids (X=30, 50, 70) that are stable in water and in the water/alcohol mixtures used to prepare the nanoinks. Further overcoating of AuNP@Cy7-C6/PEG50 % with PAH (polyallylamine hydrochloride) avoids LSPR hybridization in the dry nanoink printouts, that present two separate bands. When irradiated with laser sources near their absorption maxima, the printouts of the AuNP@Cy7-C6/PEG50 %@PAH nanoink respond on two channels, giving different temperature increases depending on the irradiation wavelengths. This enhances the potentiality of use of these nanoinks for photothermal anticounterfait printouts, making more difficult to reproduce the correct ΔT vs λirradiation output.
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A deep understanding of the interactions between micelle-like aggregates and antineoplastic drugs is paramount to control their adequate delivery. Herein, Poly(NIPAM-co-SPMA) copolymer nanocarriers were synthesized according to our previous published methodology, and the loading and release of poorly and highly water-soluble doxorubicin forms (Dox and Dox-HCl, respectively) were evaluated upon UV light irradiation and pH-variation stimuli. Capillary electrophoresis (CE) coupled to a fluorescence detector (LIF) allowed us to specifically characterize these systems and deeply study the loading and release processes. For this purpose, varying concentrations of doxorubicin were tested, and the loading/release rates were indirectly quantified thanks to the "free" doxorubicin concentration in solution. This study highlighted that Dox loading (9.4 µg/mg) was more effective than Dox-HCl loading (5.5 µg/mg). In contrast, 68 and 74% of Dox-HCl were respectively released after 2 min upon pH variation (from 7.4 to 6.0) and combined UV + pH 6.0 stimuli, while only 27% of Dox was invariably released upon application of the same stimuli. These results are coherent with the characteristics of both DoxHCl and Dox: Electrostatic interactions between Dox-HCl and the micelle-membrane structure (NIPAM) seemed predominant, while hydrophobic interactions were expected between Dox and the SP moieties at the inner part of the micelle-like aggregate, leading to different behaviors in both loading and release of the two doxorubicin forms. For doxorubicin loading concentrations higher than 3 µM, the electrophoretic profiles presented an additional peak. Thanks to CE characterizations, this peak was attributed to the formation of a complex formed between the nonaggregated copolymer and the doxorubicin molecules. This report therefore undergoes deep characterization of the dynamic formation of different micelle/drug complexes involved in the global drug-delivery behavior and therefore contributes to the development of more effective stimuli-responsive nanocarriers.
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Antineoplásicos , Micelas , Rayos Ultravioleta , Doxorrubicina/química , Sistemas de Liberación de Medicamentos/métodos , Polímeros/química , Concentración de Iones de Hidrógeno , Portadores de Fármacos/químicaRESUMEN
The possibility to shape stimulus-responsive optical polymers, especially hydrogels, by means of laser 3D printing and ablation is fostering a new concept of "smart" micro-devices that can be used for imaging, thermal stimulation, energy transducing and sensing. The composition of these polymeric blends is an essential parameter to tune their properties as actuators and/or sensing platforms and to determine the elasto-mechanical characteristics of the printed hydrogel. In light of the increasing demand for micro-devices for nanomedicine and personalized medicine, interest is growing in the combination of composite and hybrid photo-responsive materials and digital micro-/nano-manufacturing. Existing works have exploited multiphoton laser photo-polymerization to obtain fine 3D microstructures in hydrogels in an additive manufacturing approach or exploited laser ablation of preformed hydrogels to carve 3D cavities. Less often, the two approaches have been combined and active nanomaterials have been embedded in the microstructures. The aim of this review is to give a short overview of the most recent and prominent results in the field of multiphoton laser direct writing of biocompatible hydrogels that embed active nanomaterials not interfering with the writing process and endowing the biocompatible microstructures with physically or chemically activable features such as photothermal activity, chemical swelling and chemical sensing.
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Materiales Biocompatibles , Hidrogeles , Rayos Láser , Polímeros , Impresión TridimensionalRESUMEN
There is a challenging need for the development of new alternative nanostructures that can allow the coupling and/or encapsulation of therapeutic/diagnostic molecules while reducing their toxicity and improving their circulation and in-vivo targeting. Among the new materials using natural building blocks, peptides have attracted significant interest because of their simple structure, relative chemical and physical stability, diversity of sequences and forms, their easy functionalization with (bio)molecules and the possibility of synthesizing them in large quantities. A number of them have the ability to self-assemble into nanotubes, -spheres, -vesicles or -rods under mild conditions, which opens up new applications in biology and nanomedicine due to their intrinsic biocompatibility and biodegradability as well as their surface chemical reactivity via amino- and carboxyl groups. In order to obtain nanostructures suitable for biomedical applications, the structure, size, shape and surface chemistry of these nanoplatforms must be optimized. These properties depend directly on the nature and sequence of the amino acids that constitute them. It is therefore essential to control the order in which the amino acids are introduced during the synthesis of short peptide chains and to evaluate their in-vitro and in-vivo physico-chemical properties before testing them for biomedical applications. This review therefore focuses on the synthesis, functionalization and characterization of peptide sequences that can self-assemble to form nanostructures. The synthesis in batch or with new continuous flow and microflow techniques will be described and compared in terms of amino acids sequence, purification processes, functionalization or encapsulation of targeting ligands, imaging probes as well as therapeutic molecules. Their chemical and biological characterization will be presented to evaluate their purity, toxicity, biocompatibility and biodistribution, and some therapeutic properties in vitro and in vivo. Finally, their main applications in the biomedical field will be presented so as to highlight their importance and advantages over classical nanostructures.
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Materiales Biocompatibles/síntesis química , Portadores de Fármacos/síntesis química , Nanoestructuras/química , Péptidos/síntesis química , Técnicas de Síntesis en Fase Sólida/métodos , Secuencia de Aminoácidos , Animales , Materiales Biocompatibles/farmacocinética , Portadores de Fármacos/farmacocinética , Composición de Medicamentos/métodos , Humanos , Nanoestructuras/administración & dosificación , Nanoestructuras/ultraestructura , Tamaño de la Partícula , Péptidos/farmacocinética , Distribución TisularRESUMEN
Youth mentoring is theorized as a relationship-based intervention in which a strong mentor-mentee bond functions as a mediator of positive outcomes. Given evidence for the importance of a positive relationship, the current study investigated whether differences in mentors' self-reported attachment tendencies (avoidance and ambivalence), Big Five personality traits, and self-efficacy predicted match quality after one academic semester. We also tested whether mentors' experience of conflict in the relationship moderated the relation between these characteristics and match quality. Participants were college student mentors (N = 190) paired with elementary school children identified via teacher and peer reports as highly aggressive. Separate regression analyses indicated that avoidance, openness, and self-efficacy significantly predicted mentor-rated (but not child-rated) match quality in expected directions. Moderator analyses revealed a mixed pattern of results: at low levels of conflict, ambivalence was a negative predictor of match quality, whereas extraversion and agreeableness were positive predictors. At high levels of conflict, openness and conscientiousness were positive predictors of match quality, whereas agreeableness was a negative predictor. The findings suggest it is important for mentoring programs to consider mentor characteristics when screening, training, and matching mentors, particularly in relationships with children identified as aggressive.
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Agresión , Conducta Infantil , Tutoría , Apego a Objetos , Personalidad , Autoeficacia , Adolescente , Niño , Femenino , Humanos , Masculino , Encuestas y Cuestionarios , Adulto JovenRESUMEN
Antibacterial treatment is an essential issue in many diverse fields, from medical device treatments (for example prostheses coating) to food preservation. However, there is a need of novel and light-weight materials with high antibacterial efficiency (preferably due to the physical activation). Utilization of photo-thermally active nanoparticles can lead to novel and re-usable materials that can be remotely activated on-demand to thermally eradicate bacteria and mitigate biofilm formation, therefore meeting the above challenge. In this study polyvinyl alcohol (PVA) hydrogel films containing non-toxic and highly photo-thermally active Prussian blue (PB) nanoparticles were fabricated. The confocal microscopy studies indicated a uniform nanoparticle distribution and a low degree of aggregation. Upon near-infrared (NIR; 700 and 800 nm) light irradiation of PVA-PB films, the local temperature increases rapidly and reaches a plateau (up to ΔT â 78 °C), within ≈6-10 s under relatively low laser intensities, I â 0.3 W cm-2. The high and localized increase of temperature on the fabricated films resulted in an efficient antibacterial effect on Pseudomonas aeruginosa (P. aeruginosa) bacteria. In addition, the localized photo-thermal effect was also sufficient to substantially mitigate biofilms growth.
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Antibacterianos/síntesis química , Biopelículas/efectos de los fármacos , Ferrocianuros/química , Nanopartículas/química , Fototerapia/métodos , Alcohol Polivinílico/química , Ferrocianuros/farmacología , Calor , Rayos Láser , Terapia por Luz de Baja Intensidad/métodos , Alcohol Polivinílico/farmacología , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/fisiologíaRESUMEN
Microfluidic devices reproducing 3D networks are particularly valuable for nanomedicine applications such as tissue engineering and active cell sorting. There is however a gap in the possibility to measure how the flow evolves in such 3D structures. We show here that it is possible to map 3D flows in complex microchannel networks by combining wide field illumination to image correlation approaches. For this purpose, we have derived the spatiotemporal image correlation analysis of time stacks of single-plane illumination microscopy images. From the detailed analytical and numerical analysis of the resulting model, we developed a fitting method that allows us to measure, besides the in-plane velocity, the out-of-plane velocity component down to vz â 65 µm/s. We have applied this method successfully to the 3D reconstruction of flows in microchannel networks with planar and 3D ramifications. These different network architectures have been realized by exploiting the great prototyping ability of a 3D printer, whose precision can reach few tens of micrometers, coupled to poly dimethyl-siloxane soft-printing lithography.
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The use of functionalized magnetic particles is increasing because they simplify the analytical process and yield promising results in a wide range of applications. Particularly, streptavidin-coated magnetic beads offer the possibility of rapid and very efficient grafting of biomolecules. Unfortunately, current methods to monitor and compute this grafting process are cumbersome and scarce. We describe herein a simple, rapid, and reliable chemiluminescent assay we have developed to check the grafting rate of functionalized magnetic beads. The power of the assay also relies on its ability to predict the amount of ligands required to obtain a precise grafting rate. In addition, results were correlated with a more general parameter in material functionalization characterization like surface ligand density. Finally, the assay was validated for a wide variety of biotinylated biomolecule sizes, ranging from small molecules (around 200 Da) to antibodies (around 150 kDa). This approach will allow a precise quantification and prediction of the functionalization of magnetic particles that is of enormous importance for quality control in many applications.
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Mediciones Luminiscentes/normas , Magnetismo , Proteínas/química , Estreptavidina/química , Bioensayo , Biotinilación , Peroxidasa de Rábano Silvestre/química , Ligandos , Peso Molecular , Propiedades de SuperficieRESUMEN
Angiogenesis and its involved proteins, particularly Vascular Endothelial Growth Factor family (VEGFs) and VEGF receptors (VEGFRs), have been considered as a target of therapeutic interest for numerous inflammatory and vascular diseases. Acting on this biological process through interaction with VEGFs or VEGFRs has received considerable attention. Indeed, VEGFs and VEGFRs are currently targeted by drugs such as monoclonal antibodies. The feasibility of a therapeutic strategy based on blocking the VEGF/VEGFR interaction by using ligands "other-than-biologics" is also explored. To help to the discovery of new molecules, screening assays have been developed, particularly to evaluate the VEGFA/VEGFR1 interaction. Despite the therapeutic importance of VEGFB and PlGF (Placental Growth Factor), no assays have been developed to evaluate molecules against their interactions with VEGFR1. Here, we present new versatile colorimetric immunoassays to screen and evaluate the specific interaction of discovered molecules with different growth factors (VEGFA, VEGFB, PlGF) and receptors (VEGFR1, VEGFR2). These tests, based on competitive immunoassay format, will provide essential information on specificity and selectivity of molecules for their targets and will help to work on the pharmaco-modulation of molecules for targeting one specific interaction.
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Colorimetría , Inmunoensayo , Receptores de Factores de Crecimiento Endotelial Vascular/análisis , Factores de Crecimiento Endotelial Vascular/análisis , Humanos , Receptores de Factores de Crecimiento Endotelial Vascular/metabolismo , Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
In Saccharomyces cerevisiae the second messenger cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) play a central role in metabolism regulation, stress resistance and cell cycle progression. To monitor cAMP levels and PKA activity in vivo in single S. cerevisiae cells, we expressed an Epac-based FRET probe and a FRET-based A-kinase activity reporter, which were proven to be useful live-cell biosensors for cAMP levels and PKA activity in mammalian cells. Regarding detection of cAMP in single yeast cells, we show that in wild type strains the CFP/YFP fluorescence ratio increased immediately after glucose addition to derepressed cells, while no changes were observed when glucose was added to a strain that is not able to produce cAMP. In addition, we had evidence for damped oscillations in cAMP levels at least in SP1 strain. Regarding detection of PKA activity, we show that in wild type strains the FRET increased after glucose addition to derepressed cells, while no changes were observed when glucose was added to either a strain that is not able to produce cAMP or to a strain with absent PKA activity. Taken together these probes are useful to follow activation of the cAMP/PKA pathway in single yeast cells and for long times (up to one hour).
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Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Colorantes Fluorescentes/análisis , Saccharomyces cerevisiae/metabolismo , Análisis de la Célula Individual/métodos , AMP Cíclico/análisis , Proteínas Quinasas Dependientes de AMP Cíclico/análisis , Colorantes Fluorescentes/química , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/enzimologíaRESUMEN
This paper gives a critical overview of capillary electrophoresis (CE) methodologies recently developed for controlling and optimizing the synthesis of nanoparticles as well as characterizing their functionalization in terms of physicochemical properties. Thanks to their electrophoretic mobility, various parameters can be determined, such as NP size and charge distribution, ζ-potential, surface functionality, colloidal stability, grafting rates, and dissociation constants, allowing not only the complete characterization of new nanoprobes but also helping in their design and in the selection of chemical conditions for their storage and further manipulation. New strategies for the improvement of CE detection sensitivity are also described.
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Electroforesis Capilar/métodos , NanopartículasRESUMEN
Anisotropic metallic nanoparticles have been devised as powerful potential tools for in vivo imaging, photothermal therapy, and drug delivery thanks to plasmon-enhanced absorption and scattering cross sections, ease in synthesis and functionalization, and controlled cytotoxicity. The rational design of all these applications requires the characterization of the nanoparticles intracellular trafficking pathways. In this work, we exploit live-cell time-lapse confocal reflectance microscopy and image correlation in both direct and reciprocal space to investigate the intracellular transport of branched gold nanostars (GNSs). Different transport mechanisms, spanning from pure Brownian diffusion to (sub-)ballistic superdiffusion, are revealed by temporal and spatio-temporal image correlation spectroscopy on the tens-of-seconds timescale. According to these findings, combined with numerical simulations and with a Bayesian (hidden Markov model-based) analysis of single particle tracking data, we ascribe the superdiffusive, subballistic behavior characterizing the GNSs dynamics to a two-state switching between Brownian diffusion in the cytoplasm and molecular motor-mediated active transport. For the investigation of intermittent-type transport phenomena, we derive an analytical theoretical framework for Fourier-space image correlation spectroscopy (kICS). At first, we evaluate the influence of all the dynamic and kinetic parameters (the diffusion coefficient, the drift velocity, and the transition rates between the diffusive and the active transport regimes) on simulated kICS correlation functions. Then we outline a protocol for data analysis and employ it to derive whole-cell maps for each parameter underlying the GNSs intracellular dynamics. Capable of identifying even simpler transport phenomena, whether purely diffusive or ballistic, our intermittent kICS approach allows an exhaustive investigation of the dynamics of GNSs and biological macromolecules.
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Oro/química , Oro/metabolismo , Espacio Intracelular/metabolismo , Microscopía Confocal , Modelos Biológicos , Movimiento , Nanoestructuras , Supervivencia Celular , Difusión , Endocitosis , Células HeLa , Humanos , Procesamiento de Imagen Asistido por Computador , Análisis Espacio-TemporalRESUMEN
The stability of thiol bonding on the surface of star-shaped gold nanoparticles was studied as a function of temperature in water and in a set of biologically relevant conditions. The stability was evaluated by monitoring the release of a model fluorescent dye, Bodipy-thiol (BDP-SH), from gold nanostars (GNSs) cocoated with poly(ethylene glycol) thiol (PEG-SH). The increase in the BDP-SH fluorescence emission, quenched when bound to the GNSs, was exploited to this purpose. A maximum 15% dye release in aqueous solution was found when the bulk temperature of gold nanostars solutions was increased to T = 42 °C, the maximum physiological temperature. This fraction reduces 3-5% for temperatures lower than 40 °C. Similar results were found when the temperature increase was obtained by laser excitation of the near-infrared (NIR) localized surface plasmon resonance of the GNSs, which are photothermally responsive. Besides the direct impact of temperature, an increased BDP-SH release was observed upon changing the chemical composition of the solvent from pure water to phosphate-buffered saline and culture media solutions. Moreover, also a significant fraction of PEG-SH was released from the GNS surface due to the increase in temperature. We monitored it with a different approach, that is, by using a coating of α-mercapto-ω-amino PEG labeled with tetramethylrhodamine isothiocyanate on the amino group, that after heating was separated from GNS by ultracentrifugation and the released PEG was determined by spectrofluorimetric techniques on the supernatant solution. These results suggest some specific limitations in the use of the gold-thiolate bond for coating of nanomaterials with organic compounds in biological environments. These limitations come from the duration and the intensity of the thermal treatment and from the medium composition and could also be exploited in biological media to modulate the in vivo release of drugs.
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Oro/química , Nanopartículas del Metal/química , Nanoestructuras/química , Compuestos de Sulfhidrilo/química , Propiedades de SuperficieRESUMEN
A polymer complex (1P) was synthesized by binding bis(cyclometalated) Ir(ppy)2(+) fragments (ppy = 2-phenylpyridyl) to phenanthroline (phen) pendants of a poly(amidoamine) copolymer (PhenISA, in which the phen pendants involved â¼6% of the repeating units). The corresponding molecular complex [Ir(ppy)2(bap)](+) (1M, bap = 4-(butyl-4-amino)-1,10-phenanthroline) was also prepared for comparison. In water solution 1P gives nanoaggregates with a hydrodynamic diameter of 30 nm in which the lipophilic metal centers are presumed to be segregated within polymer tasks to reduce their interaction with water. Such confinement, combined with the dilution of triplet emitters along the polymer chains, led to 1P having a photoluminescence quantum yield greater than that of 1M (0.061 vs 0.034, respectively, in an aerated water solution) with a longer lifetime of the (3)MLCT excited states and a blue-shifted emission (595 nm vs 604 nm, respectively). NMR data supported segregation of the metal centers. Photoreaction of O2 with 1,5-dihydroxynaphthalene showed that 1P is able to sensitize (1)O2 generation but with half the quantum yield of 1M. Cellular uptake experiments showed that both 1M and 1P are efficient cell staining agents endowed with two-photon excitation (TPE) imaging capability. TPE microscopy at 840 nm indicated that both complexes penetrate the cellular membrane of HeLa cells, localizing in the perinuclear region. Cellular photodynamic therapy tests showed that both 1M and 1P are able to induce cell apoptosis upon exposure to Xe lamp irradiation. The fraction of apoptotic cells for 1M was higher than that for 1P (74 and 38%, respectively) 6 h after being irradiated for 5 min, but cells incubated with 1P showed much lower levels of necrosis as well as lower toxicity in the absence of irradiation. More generally, the results indicate that cell damage induced by 1M was avoided by binding the iridium sensitizers to the poly(amidoamine).
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Iridio/química , Sustancias Luminiscentes/química , Compuestos Organometálicos/química , Fotoquimioterapia , Fármacos Fotosensibilizantes/química , Poliaminas/química , Oxígeno Singlete/química , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Estabilidad de Medicamentos , Células HeLa , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Sustancias Luminiscentes/farmacología , Naftoles/química , Compuestos Organometálicos/farmacología , Oxidación-Reducción , Procesos Fotoquímicos , Fármacos Fotosensibilizantes/farmacologíaRESUMEN
A one-step synthesis of near infrared fluorescent platinum nanoclusters (PtNCs) in aqueous medium is described. The proposed optimized procedure for PtNC synthesis is rather simple, fast and it is based on the direct metal reduction with NaBH4. Bidentated thiol ligands (lipoic acid) were selected as nanoclusters stabilizers in water media. The structural characterization revealed attractive features of the PtNCs, including small size, high water solubility, near-infrared luminescence centered at 680 nm, long-term stability and the highest quantum yield in water reported so far (47%) for PtNCs. Moreover, their stability in different pH media and an ionic strength of 0.2 M NaCl was studied and no significant changes in fluorescence emission were detected. In brief, they offer a new type of fluorescent noble metal nanoprobe with a great potential to be applied in several fields, including biolabeling and imaging experiments.
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The armory of GFP mutants available to biochemists and molecular biologists is huge. Design and selection of mutants are usually driven by tailored spectroscopic properties, but some key aspects of stability, folding and dynamics of selected GFP variants still need to be elucidated. We have prepared, expressed and characterized three H148 mutants of the highly fluorescent variant GFPmut2. H148 is known to be involved in the H-bonding network surrounding the chromophore, and all the three mutants, H148G, H148R and H148K, show increased pKa values of the chromophore. Only H148G GFPmut2 (Mut2G) gave good expression and purification yields, indicating that position 148 is critical for efficient folding in vivo. The chemical denaturation of Mut2G was monitored by fluorescence emission, absorbance and far-UV circular dichroism spectroscopy. The mutation has little effect on the spectroscopic properties of the protein and on its stability in solution. However, the unfolding kinetics of the protein encapsulated in wet nanoporous silica gels, a system that allows to stabilize conformations that are poorly or only transiently populated in solution, indicate that the unfolding pathway of Mut2G is markedly different from the parent molecule. In particular, encapsulation allowed to identify an unfolding intermediate that retains a native-like secondary structure despite a destructured chromophore environment. Thus, H148 is a critical residue not only for the chromophoric and photodynamic properties, but also for the correct folding of GFP, and its substitution has great impact on expression yields and stability of the mature protein.
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Proteínas Fluorescentes Verdes/química , Histidina/química , Pliegue de Proteína , Estabilidad Proteica , Cinética , Mutación , Conformación Proteica , Estructura Secundaria de Proteína , Análisis Espectral , Relación Estructura-Actividad , TermodinámicaRESUMEN
We developed an all-optical method to measure the temperature on gold (nanorods and nanostars) and magnetite nanoparticles under near-infrared and radiofrequency excitation by monitoring the excited state lifetime of Rhodamine B that lies within =/~20 nm from the nanoparticle surface. We reached high temperature sensitivity (0.029 ± 0.001 ns/°C) and low uncertainty (±0.3 °C). Gold nanostars are =/~3 and =/~100 times more efficient than gold nanorods and magnetite nanoparticles in inducing localized hyperthermia.
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Oro/química , Nanopartículas de Magnetita/química , Nanopartículas del Metal/química , Temperatura , Fenómenos Ópticos , Tamaño de la Partícula , Propiedades de SuperficieRESUMEN
Nanostructures formed by the self-assembling peptide building blocks are attractive materials for the design of theranostic objects due to their intrinsic biocompatibility, accessible surface chemistry as well as cavitary morphology. Short peptide synthesis and modification are straightforward and give access to a great diversity of sequences, making them very versatile building blocks allowing for the design of thoroughly controlled self-assembled nanostructures. In this work, we developed a new CE-DAD-ESI-MS method to characterize short synthetic amphiphilic peptides in terms of exact sequence and purity level in the low 0.1 mg.mL-1 range, without sample treatment. This study was conducted using a model sequence, described to have pH sensitive self-assembling property. Peptide samples obtained from different synthesis processes (batch or flow, purified or not) were thus separated by capillary zone electrophoresis (CZE). The associated dual UV and MS detection mode allowed to evidence the exact sequence together with the presence of impurities, identified as truncated or non-deprotected sequences, and to quantify their relative proportion in the peptide mixture. Our results demonstrate that the developed CE-DAD-ESI-MS method could be directly applied to the characterization of crude synthetic peptide products, in parallel with the optimization of peptide synthetic pathway to obtain controlled sequences with high synthetic yield and purity, which is crucial for further design of robust peptide based self-assembled nanoarchitectures.
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Nanoestructuras , Nanomedicina Teranóstica , Electroforesis Capilar , Espectrometría de Masas , Péptidos , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
An innovative integrated paper-based microdevice was developed for protein separation by isoelectric focusing (IEF), allowing for robust design thanks to a 3D-printed holder integrating separation channel, reservoirs, and electrodes. To reach robustness and precision, the optimization focused on the holder geometry, the paper nature, the reservoir design, the IEF medium, and various focusing parameters. A well-established and stable pH gradient was obtained on a glass-fiber paper substrate with simple sponge reservoirs, and the integration of the electrodes in the holder led to a straightforward system. The separation medium composed of water/glycerol (85/15, v/v) allowed for reducing medium evaporation while being an efficient medium for most hydrophobic and hydrophilic proteins, compatible with mass spectrometry detection for further proteomics developments. To our knowledge, this is the first report of the use of glycerol solutions as a separation medium in a paper-based microdevice. Analytical performances regarding pH gradient generation, pI determination, separation efficiency, and resolution were estimated while varying the IEF experimental parameters. The overall process led to an efficient separation within 25 min. Then, this methodology was applied to a sample composed of saliva doped with proteins. A minimal matrix effect was evidenced, underscoring the practical viability of our platform. This low-cost, versatile and robust paper-based IEF microdevice opens the way to various applications, ranging from sample pre-treatment to integration in an overall proteomic-on-a-chip device.
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Glicerol , Focalización Isoeléctrica , Papel , Proteínas , Focalización Isoeléctrica/instrumentación , Focalización Isoeléctrica/métodos , Proteínas/análisis , Proteínas/aislamiento & purificación , Glicerol/química , Glicerol/análisis , Concentración de Iones de Hidrógeno , Diseño de Equipo , Humanos , Dispositivos Laboratorio en un Chip , Saliva/química , Técnicas Analíticas Microfluídicas/instrumentación , Proteómica/métodos , Interacciones Hidrofóbicas e HidrofílicasRESUMEN
Trapping of organic molecules and dyes within nanoporous matrices is of great interest for the potential creation of new materials with tailored features and, thus, different possible applications ranging from nanomedicine to material science. The understanding of the physical basis of entrapment and the spectral properties of the guest molecules within the host matrix is an essential prerequisite for the design and control of the properties of these materials. In this work, we show that a mesoporous silica xerogel can efficiently trap the dye thioflavin T (ThT, a molecule used as a marker of amyloid fibrils and with potential drug benefits), sequestering it from an aqueous solution and producing a highly fluorescent material with a ThT quantum yield 1500 times greater than that of the free molecule. The study of spectroscopical properties of this system and the comparison with fluorescence of an uncharged analogue of ThT give indications about the mechanism responsible for the fluorescence switching-on of ThT molecules during their uptaking into the glass. Diffusion and nanocapillarity are responsible for ThT absorption, whereas electrostatic interaction between positive ThT molecules and negative dangling ≡SiO groups covering the pore surfaces causes the immobilization of ThT molecules inside the pores and the enhancement of its fluorescence, in line with the molecular rotor model proposed for this dye. We also show that entrapment efficiency and kinetics can be tuned by varying the electrostatic properties of the dye and/or the matrix.