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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
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
Here, we propose a recyclable approach using acrylonitrile-butadiene-styrene (ABS) residues from additive manufacturing in combination with low-cost and accessible graphite flakes as a novel and potential mixture for creating a conductive paste. The graphite particles were successfully incorporated in the recycled thermoplastic composite when solubilized with acetone and the mixture demonstrated greater adherence to different substrates, among which cellulose-based material made possible the construction of a paper-based electrochemical sensor (PES). The morphological, structural, and electrochemical characterizations of the recycled electrode material were demonstrated to be similar to those of the traditional carbon-based surfaces. Faradaic responses based on redox probe activity ([Fe(CN)6]3-/4-) exhibited well-defined peak currents and diffusional mass transfer as a quasi-reversible system (96 ± 5 mV) with a fast heterogeneous rate constant value of 2 × 10-3 cm s-1. To improve the electrode electrochemical properties, both the PES and the classical 3D-printed electrode surfaces were modified with a combination of multiwalled carbon nanotubes (MWCNTs), graphene oxide (GO), and copper. Both electrode surfaces demonstrated the suitable oxidation of nitrite at 0.6 and 0.5 V vs Ag, respectively. The calculated analytical sensitivities for PES and 3D-printed electrodes were 0.005 and 0.002 µA/(µmol L-1), respectively. The proposed PES was applied for the indirect amperometric analysis of S-nitroso-cysteine (CysNO) in serum samples via nitrite quantitation, demonstrating a limit of detection of 4.1 µmol L-1, with statistically similar values when compared to quantitative analysis of the same samples by spectrophotometry (paired t test, 95% confidence limit). The evaluated electroanalytical approach exhibited linear behavior for nitrite in the concentration range between 10 and 125 µmol L-1, which is suitable for realizing clinical diagnosis involving Parkinson's disease, for example. This proof of concept shows the great promise of this recyclable strategy combining ABS residues and conductive particles in the context of green chemical protocols for constructing disposable sensors.
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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
A low-cost and straightforward hybrid NOA (Norland optical adhesive) 81-glass microchip electrophoresis device was designed and developed for protein separation using indirect fluorescence detection. This new microchip was first characterized in terms of surface charge density via electroosmotic mobility measurement and stability over time. A systematic determination of the electroosmotic mobility (µeo ) over a wide pH range (2-10) and at various ionic strengths (20-50 mM) was developed for the first time via the neutral marker approach in an original simple frontal methodology. The evolution of µeo was proved consistent with the silanol and thiol functions arising from the glass and the NOA materials, respectively. The repeatability and reproducibility of the measurements on different microchips (RSD < 14%) and within 15 days (less than 5% decrease) were successfully demonstrated. The microchip was then applied for the efficient electrophoretic separation of proteins in a zonal mode coupled with indirect fluorescence detection, which is, to our knowledge, the first proof of concept of capillary zone electrophoresis in this hybrid microsystem.
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Electroforesis por Microchip , Electroforesis Capilar/métodos , Electroforesis por Microchip/métodos , Vidrio/química , Proteínas/análisis , Reproducibilidad de los Resultados , Compuestos de SulfhidriloRESUMEN
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
The affinity between functional nanoparticles (NPs) and proteins could determine the efficacy of nanoprobes, nanosensors, nanocarriers, and many other devices for biomedical applications. Therefore, it is necessary to develop analytical strategies to accurately evaluate the magnitude of these protein corona interactions in physiological media. In this work, different electrokinetic strategies were implemented to accurately determine the interactions between PEGylated ZnGa1.995Cr0.005O4 persistent luminescent NPs (ZGO-PEG) and two important serum proteins: human serum albumin (HSA), the most abundant serum protein, and apolipoprotein-E (ApoE), associated with the active transport of NPs through the blood-brain barrier. Firstly, the injection of ZGO-PEG in a background electrolyte (BGE) containing individual proteins allowed an affinity study to separately characterize each NP-protein system. Then, the same procedure was applied in a buffer containing a mixture of the two proteins at different molar ratios. Finally, the NPs were pre-incubated with one protein and thereafter electrokinetically separated in a BGE containing the second protein. These analytical strategies revealed the mechanisms (comparative, cooperative or competitive systems) and the magnitude of their interactions, resulting in all cases in notably higher affinity and stability between ZGO-PEG and ApoE (Ka = 1.96 ± 0.25 × 1010 M-M) compared to HSA (Ka = 4.60 ± 0.41 × 106 M-M). For the first time, the inter-protein ApoE/HSA interactions with ZGO-PEG were also demonstrated, highlighting the formation of a ternary ZGO-PEG/ApoE/HSA nanocomplex. These results open the way for a deeper understanding of the protein corona formation, and the development of versatile optical imaging applications for ZGO-PEG and other systemically delivered nanoprobes ideally vectorized to the brain.
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Nanopartículas , Corona de Proteínas , Albúminas , Apolipoproteínas , Apolipoproteínas E , Humanos , LuminiscenciaRESUMEN
Waste printed circuit boards are a major source of strategic materials such as platinum group metals since they are used for the fabrication of technological devices, such as hard drive discs, capacitors, and diodes. Because of the high cost of platinum, palladium, and gold (> 25 k/kg), an economic and environmental challenge is their recycling from printed circuit boards that represent around 2% weight of electronic equipment. Hydrometallurgical treatments allow the recovery of these metals in solution, with a high recovery rate for a leaching liquor made of thiourea in hydrochloric acid. So as to develop an efficient recycling process from this leach liquor, one requires the speciation of these strategic metals, as well as their extraction and quantitation in the mixture. For this purpose, platinum, palladium, and gold were dissolved in model leach liquors made of hydrochloric acid and thiourea at low concentration. The identification of metal complexes was determined as a function of thiourea concentration (between 10 µmol/L and 10 mmol/L) by the combination of UV-visible spectrometry, cyclic voltammetry, and for the first time capillary electrophoresis. The electrokinetic method was then applied for the quantitation of trace metal analyses in leach samples from waste printed circuit boards reprocessing, demonstrating its applicability for industrializable recycling applications. Graphical abstract.
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Capillary zone electrophoresis (CZE) complemented with Taylor Dispersion Analysis-CE (TDA-CE) was developed to physicochemically characterize phthalocyanine-capped core/shell/shell quantum dots (QDs) at various pH and ionic strengths. An LED-induced fluorescence detector was used to specifically detect the QDs. The electropherograms and taylorgrams allowed calculating the phthalocyanine-QDs (Pc-QDs) ζ-potential and size, respectively, and determining the experimental conditions for colloidal stability. This methodology allowed evidencing either a colloidal stability or an aggregation state according to the background electrolytes nature. The calculated ζ-potential values of Pc-QDs decreased when ionic strength increased, being well correlated with the aggregation of the nanoconjugates at elevated salt concentrations. For the same reason, the hydrodynamic diameter of Pc-QDs increased with increasing background electrolyte ionic strength. The use of electrokinetic methodologies has provided insights into the colloidal stability of the photosensitizer-functionalized QDs in physiologically relevant solutions and, thereby, its usefulness for improving their design and applications for photodynamic therapy.
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Electroforesis Capilar , Indoles , Puntos Cuánticos/química , Fluorescencia , Concentración de Iones de Hidrógeno , Isoindoles , Concentración Osmolar , Fármacos FotosensibilizantesRESUMEN
The self-assembly of peptide nanotubes (PNTs) depends on the structure and chemistry of cyclic peptide (CP) monomers, impacting on their properties, which makes the choice of their monomers and their characterization a high challenge. For this purpose, we developed for the first time a capillary electrophoresis coupled to electrospray ionization mass spectrometry (CE-ESI-MS) methodology and characterized a set of eight original CP sequences of 8, 10, and 12 D,L-α-alternate amino acids with a controlled internal diameter (from 7 to 13 Å) and various properties (diameter, global surface charge, hydrophobicity). This new CE-ESI-MS methodology allows verifying the structure, the purity, as well as the stability (when stored during several months) of interesting potential precursors for PNTs that could be employed as nanoplatforms in diagnostics or pseudo sieving tools for separation purposes.
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Aminoácidos/química , Electroforesis Capilar/métodos , Nanotubos/química , Péptidos Cíclicos/química , Espectrometría de Masa por Ionización de Electrospray/métodos , Secuencia de Aminoácidos , Interacciones Hidrofóbicas e Hidrofílicas , Electricidad EstáticaRESUMEN
S-nitrosothiols (RSNOs) are very important biomolecules that play crucial roles in many physiological and physiopathological processes. They act as NO-donors and are candidates for future medicines. Their identification and quantitation are therefore important for biomedical applications. One, two or more RSNOs can then be combined to design a drug and therefore, the quantification of each is important to establish an acceptable quality control process. Till date, miniaturized devices have been used to detect RSNOs based on their total quantitation without a preceding separation step. This study reports on an original and integrated microdevice allowing for the successive electrokinetic separation of low molecular weight RSNOs, their decomposition under metal catalysis, and their quantitation by amperometric detection of the produced nitrite in the end-channel arrangement, leading to their quantitation in a single run. For this purpose, a commercial SU-8/Pyrex microfluidic system was coupled to a portable and wireless potentiostat. Different operating and running parameters were optimized to achieve the best analytical data, allowing for an LOD equal to 20 µM. The simultaneous separation of S-nitrosoglutathione and S-nitrosocysteine was successfully obtained within 75 s. The proposed methodology using SU-8/Pyrex microfluidic devices opens new possibilities to investigate future drug candidates for NO-donors.
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Cisteína/análogos & derivados , Dispositivos Laboratorio en un Chip , Técnicas Analíticas Microfluídicas/métodos , S-Nitrosoglutatión/análisis , S-Nitrosotioles/análisis , Catálisis , Cobre/química , Cisteína/análisis , Cisteína/síntesis química , Cisteína/química , Técnicas Electroquímicas/instrumentación , Técnicas Electroquímicas/métodos , Límite de Detección , Técnicas Analíticas Microfluídicas/instrumentación , S-Nitrosoglutatión/síntesis química , S-Nitrosoglutatión/química , S-Nitrosotioles/síntesis química , S-Nitrosotioles/químicaRESUMEN
In the last decades, fluorescent quantum dots (QDs) have appeared as high-performance biological fluorescent nanoprobes and have been explored for a variety of biomedical optical imaging applications. However, many central challenges still exist concerning the control of the surface chemistry to ensure high biocompatibility, low toxicity, antifouling, and specific active targeting properties. Regarding in vivo applications, circulation time and clearance of the nanoprobe are also key parameters to control the design and characterization of new optical imaging agents. Herein, the complete design and characterization of a peptide-near-infrared-QD-based nanoprobe for biomedical optical imaging is presented from the synthesis of the QDs and the zwitterionic-azide copolymer ligand, enabling a bio-orthogonal coupling, till the final in vivo test through all the characterization steps. The developed nanoprobes show high fluorescence emission, controlled grafting rate, low toxicity, in vitro active specific targeting, and in vivo long circulating blood time. This is, to our knowledge, the first report characterizing the in vivo circulation kinetics and tumor accumulation of targeted zwitterionic QDs.
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Puntos Cuánticos , Humanos , Neoplasias , Imagen Óptica , PéptidosRESUMEN
Nanoparticles (NPs) play an increasingly important role in the development of new biosensors, contrast agents for biomedical imaging and targeted therapy vectors thanks to their unique properties as well as their good detection sensitivity. However, a current challenge in developing such NPs is to ensure their biocompatibility, biodistribution, bioreactivity and in vivo stability. In the biomedical field, the adsorption of plasmatic proteins on the surface of NPs impacts on their circulation time in blood, degradation, biodistribution, accessibility, the efficiency of possible targeting agents on their surface, and their cellular uptake. NP surface passivation is therefore a very crucial challenge in biomedicine. We developed herein for the first time an electrokinetic Hummel-Dreyer method to quantitatively characterize the formation of protein corona on the surface of NPs. This strategy was designed and optimized to evaluate the non specific binding of bovine serum albumin with the recently discovered PEG-functionalized ZnGa1.995Cr0.005O4 persistent luminescence NPs developed for in vivo biological imaging. The binding strength and the number of binding sites were determined at different ionic strengths. This methodology opens the way to an easy, low sample- and low time-consuming evaluation of the impact of NP surface modification on protein-corona formation and therefore on their potential for various bio-medical applications.
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Luminiscencia , Nanopartículas/química , Corona de Proteínas/química , Sitios de Unión , Electroforesis Capilar , Albúmina Sérica Bovina/químicaRESUMEN
Persistent luminescence nanoparticles made of ZnGa1.995Cr0.005O4 (ZGO-NPs) are innovative nanomaterials that emit photons during long periods of time after the end of the excitation, allowing their use as diagnosis probes for in vivo optical imaging. During the excitation process, a part of the energy is stored in traps to further emit photons over long time. However, we observed in this study that some of the energy reduces molecular oxygen to produce reactive oxygen species (ROS). Following this observation, theoxidative stress induction and cytotoxic effects of these NPs were investigated on human breast cancer cells. The results indicate that ROS production was stimulated by exposition of the hydroxylated ZGO-NPs to UV or visible light, and the oxidative stress induced in cells after internalization can be directly correlated to their dose-dependent inhibition of cell viability. On the contrary, PEGylated ZGONPs were not uptaken by cells and have no effect on the production of ROS. Thus, the cell viability was not altered by these nanoparticles. This study reveals the importance of considering light irradiation and surface coating of luminescent nanoparticles toxicity which open new perspectives for their use in photodynamic therapy.
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Luz , Nanopartículas/efectos de la radiación , Especies Reactivas de Oxígeno/metabolismo , Muerte Celular , Línea Celular Tumoral , Humanos , Luminiscencia , Neoplasias/tratamiento farmacológicoRESUMEN
The ZnGa1.995Cr0.005O4 persistent luminescence nanoparticles offer the promise of revolutionary tools for biological imaging with applications such as cell tracking or tumor detection. They can be re-excited through living tissues by visible photons, allowing observations without any time constraints and avoiding the undesirable auto-fluorescence signals observed when fluorescent probes are used. Despite all these advantages, their uses demand extensive toxicological evaluation and control. With this purpose, mice were injected with a single intravenous administration of hydroxylated or PEGylated persistent luminescence nanoparticles at different concentrations and then a set of standard tests were carried out 1day, 1 month and 6 months after the administration. High concentrations of hydroxylated nanoparticles generate structural alterations at histology level, endoplasmic reticulum damage and oxidative stress in liver, as well as rising in white blood cells counts. A mechanism involving the endoplasmic reticulum damage could be the responsible of the observed injuries in case of ZGO-OH. On the contrary, no toxicological effects related to PEGylated nanoprobes treatment were noted during our in vivo experiments, denoting the protective effect of PEG-functionalization and thereby, their potential as biocompatible in vivo diagnostic probes.
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Cromo/toxicidad , Nanopartículas/toxicidad , Óxidos/toxicidad , Zinc/toxicidad , Animales , Recuento de Células Sanguíneas , Ensayo Cometa , Galio/toxicidad , Hidroxilación , Inyecciones Intravenosas , Riñón/efectos de los fármacos , Hígado/efectos de los fármacos , Hígado/patología , Hígado/ultraestructura , Luminiscencia , Pulmón/efectos de los fármacos , Masculino , Ratones Endogámicos BALB C , Óxido Nítrico/metabolismo , Polietilenglicoles/química , Bazo/efectos de los fármacos , Bazo/ultraestructuraRESUMEN
There is a great demand for integrating sample treatment into µTASs. In this context, we developed a new sol-gel phase for extraction of trace compounds in complex matrices. For this purpose, the incorporation of aptamers in silica-based gel within PDMS/glass microfluidic channels was performed for the first time by a one-step sol-gel process. The effective gel attachment onto microchannel walls and aptamer incorporation in the polymerized gel were evaluated using fluorescence microscopy. A good gel stability and aptamer incorporation inside the microchannel was demonstrated upon rinsing and over storage time. The ability of gel-encapsulated aptamers to interact with its specific target (either sulforhodamine B as model fluorescent target, or diclofenac, a pain killer drug) was assessed too. The binding capacity of entrapped aptamers was quantified (in the micromolar range) and the selectivity of the interaction was evidenced. Preservation of aptamers binding affinity to target molecules was therefore demonstrated. Dissociation constant of the aptamer-target complex and interaction selectivity were evaluated similar to those in bulk solution. This opens the way to new selective on-chip SPE techniques for sample pretreatment.
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Aptámeros de Nucleótidos/análisis , Dispositivos Laboratorio en un Chip , Microfluídica/métodos , Gel de Sílice/química , Analgésicos/química , Antiinflamatorios no Esteroideos/química , Cromatografía de Afinidad/métodos , Diclofenaco/química , Colorantes Fluorescentes/química , Humanos , Microfluídica/instrumentación , Transición de Fase , Rodaminas/química , Sensibilidad y Especificidad , Contaminantes Químicos del Agua/químicaRESUMEN
In this work, we characterized different phtalocyanine-capped core/shell/shell quantum dots (QDs) in terms of stability, ζ-potential, and size at various pH and ionic strengths, by means of capillary electrophoresis (CE), and compared these results to the ones obtained by laser Doppler electrophoresis (LDE) and dynamic light scattering (DLS). The effect of the phthalocyanine metallic center (Zn, Al, or In), the number (one or four), and nature of substituents (carboxyphenoxy- or sulfonated-) of functionalization on the phthalocyanine physicochemical properties were evaluated. Whereas QDs capped with zinc mono-carboxyphenoxy-phtalocyanine (ZnMCPPc-QDs) remained aggregated in the whole analyzed pH range, even at low ionic strength, QDs capped with zinc tetracarboxyphenoxy phtalocyanine (ZnTPPc-QDs) were easily dispersed in buffers at pH equal to or higher than 7.4. QDs capped with aluminum tetrasulfonated phthalocyanine (AlTSPPc-QDs) and indium tetracarboxyphenoxy phthalocyanines (InTCPPc-QDs) were stable in aqueous suspension only at pH higher than 9.0 due to the presence of functional groups bound to the metallic center of the phthalocyanine. The ζ-potential values determined by CE for all the samples decreased when ionic strength increased, being well correlated with the aggregation of the nanoconjugates at elevated salt concentrations. The use of electrokinetic methodologies has provided insights into the colloidal stability of the photosensitizer-functionalized QDs in physiological relevant solutions and thereby, its usefulness for improving their design and applications for photodynamic therapy. Graphical Abstract Schematic illustration of the phthalocyanine capped QDs nanoconjugates and the capillary electrophoresis methods applied for size and ζ-potential characterization.
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Indoles/química , Puntos Cuánticos/química , Dispersión Dinámica de Luz/métodos , Electroforesis Capilar/métodos , Indio/química , Isoindoles , Rayos Láser , Metales/química , Compuestos Organometálicos/química , Concentración Osmolar , Tamaño de la Partícula , Electricidad Estática , Zinc/químicaRESUMEN
A disposable microfluidic paper-based analytical device (µPAD) was developed to easily analyse different S-nitrosothiols (RSNOs) through colorimetric measurements. RSNOs are carriers of nitric oxide (NO) that play several physiological and physiopathological roles. The quantification of RSNOs relies on their decomposition using several protocols and the colorimetric detection of the final product, NO or nitrite. µPADs were fabricated by wax printing technology in a geometry containing one central zone for the sample inlet and eight circular detection zones interconnected by microfluidic channels for decomposition and posterior detection of decayed products. Different decomposition protocols including mercuric ions and light (UV, visible, and infrared) were tested on µPADs. For this purpose, a 3D printed holder was coupled with µPADs to easily design a simultaneous decomposition procedure using different light sources. The Griess reagent was added to detect NO and nitrite produced by the different decomposition methods. µPADs were then scanned using a flat board scanner and calibration curves based on color intensity were plotted. The limit of detection (LOD) values achieved for nitrite (used as a reference compound) and S-nitrosoglutathione (GSNO) using mercuric decomposition were 3 and 4 µM, respectively. The LOD reported herein for nitrite is considered among the lowest LODs already reported for this compound using µPADs. The results also show that low-molecular-weight RSNO, namely S-nitrosocysteine, decomposes more easily than high-molecular-weight RSNOs with light. As a proof of concept, RSNOs in human plasma were successfully detected on µPADs. For this purpose, a preliminary treatment step was optimized and the presence of high-molecular-weight (HMW) RSNOs was evidenced in the available plasma samples. The concentrations of HMW-RSNOs and nitrite in the various samples ranged from 5 to 16 µM and from 37 to 58 µM, respectively.
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Capillary isoelectric focusing (CIEF) is a high-resolution technique for the separation of ampholytes, such as proteins, according to their isoelectric point. CIEF coupled online with MS is regarded as a promising alternative to 2-D PAGE for fast proteome analysis with high-resolving capabilities and enhanced structural information without the drawbacks of conventional slab-gel electrophoresis. However, online coupling has been rarely described, as it presents some difficulties. A new methodology for the online coupling of CIEF with electrospray ionization mass spectrometry (ESI-MS) has been developed in glycerol-water media. This new integrated methodology provides a mean for the characterization of a large number of hydrophilic and hydrophobic proteins.
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Focalización Isoeléctrica/métodos , Proteínas/aislamiento & purificación , Espectrometría de Masa por Ionización de Electrospray/métodos , Glicerol/química , Concentración de Iones de Hidrógeno , Proteínas/química , Agua/químicaRESUMEN
The self-assembly of peptide nanotubes (PNTs) depends on the structure and chemistry of cyclic peptide (CP) monomers, having an impact on their properties, making the choice of their monomers and their characterization a great challenge. We synthesized for the first time a new set of eight original CP sequences of 8, 10, and 12 d,l-α-alternate amino acids with a controlled internal diameter from 7 to 13 Å. They present various properties (e.g., diameter, global surface charge, hydrophobicity) that can open the way to new applications. Their structure and purity were determined thanks to a capillary electrophoresis coupled to electrospray ionization mass spectrometry (CE-ESI-MS) methodology developed for the first time for this purpose. The CPs were successfully separated in a basic hydro-organic background electrolyte (BGE, pH 8.0, H2O/EtOH 50:50, v/v) and analyzed in MS positive mode. The effect of CP structure on electrophoretic mobility was studied, and the mass spectra were deeply analyzed. This methodology allowed verifying their purity and the absence of linear peptide precursors as well as their stability when stored over several months. Therefore, we have developed a new CE-ESI-MS methodology for the structure and purity control of interesting potential precursors for PNTs that could be employed as nanoplatforms in diagnostics or as pseudo sieving tools for separative purposes.