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Supramolecular hydrogels play a pivotal role in many fields of biomedical research, including emerging applications in designing advanced tools for point-of-care testing, clinical diagnostics, and lab-on-chip analysis. This review outlines the growing relevance of supramolecular hydrogels in biosensing and bioassay devices, highlighting recent advancements that deliver increased sensitivity, real-time monitoring, and multiplexing capabilities through the distinctive properties of these nanomaterials. Furthermore, the exploration extends to additional applications, such as using hydrogels as three-dimensional matrices for cell-based assays.
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Polyamidoamine (PAMAM) dendrimers are among the most studied cationic polymers as non-viral gene delivery vectors. However, an "ideal" PAMAM-based gene delivery vector is still missing due to the high manufacturing costs and non-negligible cytotoxicity associated with the use of high-generation dendrimers, whereas low-generation dendrimers are far from displaying efficient gene transfection. In order to cover this gap in the literature, in this study, we propose the functionalization of the outer primary amines of PAMAM G2 and PAMAM G4 with building blocks bearing fluorinated moieties along with a guanidino functional group. We have designed and synthetized two fluorinated arginine (Arg)-based Michael acceptors which were straightforwardly "clicked" to PAMAM dendrimers without the need for coupling reagents and/or catalysts. The obtained conjugates, in particular, derivative 1 formed starting from the low-cost PAMAM G2 and a building block bearing two trifluoromethyl groups, were able to efficiently complex plasmid DNA, had negligible cytotoxicity, and showed improved gene transfection efficiency as compared to undecorated PAMAM dendrimers and a corresponding unfluorinated PAMAM-Arg derivative, with derivative 1 being two orders of magnitude more efficient than the gold standard branched polyethylenimine, bPEI, 25 kDa. These results highlight the importance of the presence of trifluoromethyl moieties for both gene transfection and a possible future application in 19F magnetic resonance imaging.
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Dendrímeros , Transfección , Técnicas de Transferencia de Gen , Terapia GenéticaRESUMEN
The activity profile of many biologically relevant proteins and peptides often relies on a precise 3D structural organization. In this context, disulfide bonds are natural covalent constraints that play a key role in driving and stabilizing the folding pattern of these molecules. Despite its prominent significance as structural motif, the disulfide bond itself is inherently unstable under physiological conditions, posing a major limit to the use and development of disulfide-rich peptides and proteins as molecular tools and drug lead compounds. To tackle this restriction, disulfide engineering with stable functional analogues has arisen a considerable interest. Here, the most popular approaches to disulfide replacement are reviewed and discussed with particular emphasis on advantages and limitations under both functional and synthetic perspectives.
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The generation of robust analytical data using microarray platforms strictly relies on optimal ligand-target interaction at the sensor surface, which, in turn, is inherently bound to the correct immobilization scheme of the interrogated bioprobes. In the present work, we performed a rigorous comparative analysis of the impact of peptide ligands immobilization strategy in the screening of Burkholderia cepacia complex (BCC) infections in patients affected by cystic fibrosis (CF). We generated arrays of previously validated Burkholderia derived peptide probes that were selectively oriented on polymeric coatings by means of different click-type reactions including thiol maleimide, copper-catalyzed azide-alkyne cycloaddition (CuAAC), and strain-promoted azide-alkyne cycloaddition (SPAAC). We compared immobilization efficiency among the different chemoselective reactions, and we evaluated diagnostic performances at a statistically significant level, also in contrast to random immobilization strategies. Our findings clearly support the favorable role of correct bioprobe orientation in discriminating seronegative from infected individuals and, in the last analysis, in generating more-reliable and more-reproducible data. Spacing biomolecules from the sensor surface by means of small hydrophilic linkers also positively affects the analytical performance and leads to increased statistical significance of data. Overall, all of the click immobilization strategies that were considered displayed a good efficiency. Interestingly, SPAAC-mediated conjugation using DBCO cyclooctyne for some peptides resulted in sequence-dependent autofluorescence in the Cy5 emission range wavelength, which could be circumvented by using a different fluorescence detection channel. On the basis of our results, we critically discuss the immobilization parameters that need to be carefully considered for peptide ligand immobilization purposes.
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Proteínas Inmovilizadas/química , Péptidos/química , Polímeros/química , Análisis por Matrices de Proteínas , Alquinos/química , Secuencia de Aminoácidos , Azidas/química , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Complejo Burkholderia cepacia/fisiología , Catálisis , Química Clic , Cobre/química , Reacción de Cicloadición , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Proteínas Inmovilizadas/metabolismo , Modelos Moleculares , Péptidos/metabolismo , Conformación Proteica , Especificidad por SustratoRESUMEN
In this paper, we report on the postpolymerization modification (PPM) of a polymer to introduce new functionalities that enable click chemistry reactions for microarray applications. The parent polymer, named copoly(DMA-NAS-MAPS), is composed of N,N-dimethylacrylamide (DMA), a monomer that self-adsorbs onto different materials through weak interactions such as hydrogen bonding or van der Waals forces, 3-(trimethoxysilyl)propyl methacrylate (MAPS) that strengthens the stability of the coating through the formation of covalent bonds with siloxane groups on the surface to be coated, and N-acryloyloxysuccinimide (NAS), an active ester group, highly reactive toward nucleophiles, which enables bioprobe immobilization. This copolymer has been widely exploited to coat surfaces for microarray applications but exhibits some limitations because of the potential hydrolysis of the active ester (NHS ester). The degradation of the NHS ester hampers the use of this coating in some situations, for example, when probe immobilization cannot be accomplished through a microspotting situation, but in large volumes, for example, in microchannel derivatization or micro-/nanoparticle functionalization. To overcome the limitations of NHS esters, we have developed a family of polymers that originate from the common copolymer precursor, by reacting the active ester contained in the polymer chain with a bifunctional amine. In particular, the functional groups introduced in the polymer using PPM enable click chemistry reactions such as azide/alkyne or thiol/maleimide "click" reactions, with suitably modified biomolecules. The advantages of such reactions are quantitative yields, orthogonality of functional groups, and insensitivity of the reaction to pH. The new click functionalities, inserted with quantitative yields, improve the stability of the coating, enabling the attachment of biomolecules directly from a solution and avoiding the spotting of reduced volumes (pL) of probes. Finally, we have demonstrated the applicability of the click surfaces in a highly effective solid-phase PCR for the genotyping of the G12D KRAS mutation.
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Recently, membrane devices and processes have been applied for the separation and concentration of subcellular components such as extracellular vesicles (EVs), which play a diagnostic and therapeutic role in many pathological conditions. However, the separation and isolation of specific EV populations from other components found in biological fluids is still challenging. Here, we developed a peptide-functionalized hollow fiber (HF) membrane module to achieve the separation and enrichment of highly pure EVs derived from the culture media of human cardiac progenitor cells. The strategy is based on the functionalization of PSf HF membrane module with BPt, a peptide sequence able to bind nanovesicles characterized by highly curved membranes. HF membranes were modified by a nanometric coating with a copoly azide polymer to limit non-specific interactions and to enable the conjugation with peptide ligand by click chemistry reaction. The BPt-functionalized module was integrated into a TFF process to facilitate the design, rationalization, and optimization of EV isolation. This integration combined size-based transport of species with specific membrane sensing ligands. The TFF integrated BPt-functionalized membrane module demonstrated the ability to selectively capture EVs with diameter < 200 nm into the lumen of fibers while effectively removing contaminants such as albumin. The captured and released EVs contain the common markers including CD63, CD81, CD9 and syntenin-1. Moreover, they maintained a round shape morphology and structural integrity highlighting that this approach enables EVs concentration and purification with low shear stress. Additionally, it achieved the removal of contaminants such as albumin with high reliability and reproducibility, reaching a removal of 93%.
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Vesículas Extracelulares , Péptidos , Humanos , Vesículas Extracelulares/química , Péptidos/química , Péptidos/aislamiento & purificación , Membranas Artificiales , Tamaño de la Partícula , Propiedades de SuperficieRESUMEN
Extracellular vesicles (EVs), crucial mediators of cell-to-cell communication, hold significant diagnostic potential due to their ability to concentrate protein biomarkers in bodily fluids. However, challenges in isolating EVs from biological specimens hinder their widespread use. The preferred strategy involves direct analysis, integrating isolation and analysis solutions, with immunoaffinity methods currently dominating. Yet, the heterogeneous nature of EVs poses challenges, as proposed markers may not be as universally present as thought, raising concerns about biomarker screening reliability. This issue extends to EV-mimics, where conventional methods may lack applicability. Addressing these challenges, the study reports on Membrane Sensing Peptides (MSP) as pan-vesicular affinity ligands for both EVs and their non-canonical analogs, streamlining capture and phenotyping through Single Molecule Array (SiMoA). MSP ligands enable direct analysis of circulating EVs, eliminating the need for prior isolation. Demonstrating clinical translation, MSP technology detects an EV-associated epitope signature in serum and plasma, distinguishing myocardial infarction from stable angina. Additionally, MSP allow analysis of tetraspanin-lacking Red Blood Cell-derived EVs, overcoming limitations associated with antibody-based methods. Overall, the work underlines the value of MSP as complementary tools to antibodies, advancing EV analysis for clinical diagnostics and beyond, and marking the first-ever peptide-based application in SiMoA technology.
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Biomarcadores , Vesículas Extracelulares , Péptidos , Vesículas Extracelulares/metabolismo , Humanos , Péptidos/metabolismo , Biomarcadores/metabolismoRESUMEN
Analytical platforms for small extracellular vesicle (sEV) high-throughput analysis are highly desirable. These bionanoparticles present fairly distinctive lipid membrane features including high curvature, lipid-packing defects, and a relative abundance in lipids. sEV membrane could be considered as a "universal" marker, complementary or alternative to traditional surface-associated proteins. Here, we describe the use of membrane-sensing peptides as a new, highly efficient ligand to directly integrate sEV capturing and analysis on a microarray platform.
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Vesículas Extracelulares , Péptidos , Vesículas Extracelulares/metabolismo , Ligandos , Lípidos , Proteínas de la Membrana/metabolismo , Membranas/metabolismo , Péptidos/metabolismoRESUMEN
Recent advances in biosensing analytical platforms have brought relevant outcomes for novel diagnostic and therapy-oriented applications. In this context, 3D droplet microarrays, where hydrogels are used as matrices to stably entrap biomolecules onto analytical surfaces, potentially provide relevant advantages over conventional 2D assays, such as increased loading capacity, lower nonspecific binding, and enhanced signal-to-noise ratio. Here, we describe a hybrid hydrogel composed of a self-assembling peptide and commercial agarose (AG) as a suitable matrix for 3D microarray bioassays. The hybrid hydrogel is printable and self-adhesive and allows analyte diffusion. As a showcase example, we describe its application in a diagnostic immunoassay for the detection of SARS-CoV-2 infection.
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COVID-19 , Hidrogeles , COVID-19/diagnóstico , Humanos , Hidrogeles/química , Inmunoensayo , Péptidos/química , Cementos de Resina , SARS-CoV-2 , SefarosaRESUMEN
In SARS-CoV-2 pandemic scenario, the identification of rapid methods to detect antibodies against coronavirus has been a wide and urgent issue. Epitope mapping on peptide microarrays is a rapid way to identify sequences with a high immunoreactivity. The process begins with a proteome-wide screening, based on immune affinity; the use of a high-density microarray is followed by a validation phase, where a restricted panel of probes is tested using peptide microarrays; peptide sequences are immobilized through a click-based strategy.COVID-19-positive sera are tested and immuno-domains regions are identified on SARS-CoV-2 spike (S), nucleocapsid (N) protein, and Orf1ab polyprotein. An epitope on N protein (region 155-171) provided good diagnostic performance in discriminating COVID-19-positive vs. healthy individuals. Using this sequence, 92% sensitivity and 100% specificity are reached for IgG detection in COVID-19 samples, and no cross-reactivity with common cold coronaviruses is detected. Overall, epitope 155-171 from N protein represents a promising candidate for further development and rapid implementation in serological tests.
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COVID-19 , SARS-CoV-2 , Anticuerpos Antivirales , COVID-19/diagnóstico , Mapeo Epitopo , Epítopos , Humanos , Inmunidad , Inmunoglobulina G , Poliproteínas , Proteoma , SARS-CoV-2/genética , Glicoproteína de la Espiga del CoronavirusRESUMEN
Introduction: One main limitation in biomarker studies using EVs is the lack of a suitable isolation method rendering high yield and purity samples in a quick and easily standardized procedure. Here we report an affinity isolation method with a membrane-sensing peptide (MSP) derived from bradykinin. Methods: We designed a protocol based on agarose beads carrying cation chelates to specifically bind to the 6His-tagged membrane-sensing peptide. This approach presents several advantages: 1) cation-carrying agaroses are widely used and standardized for His-tagged protein isolation, 2) the affinity protocol can be performed in small volumes, feasible and manageable for clinical routine and 3) elution with imidazole or EDTA allows a gentle and easy recovery without EV damage, facilitating subsequent characterization and functional analyses. Results: The optimized final procedure incubates 0.5 mg of peptide for 10 min with 10 µL of Long-arm Cobalt agarose before an overnight incubation with concentrated cell conditioned medium. EV downstream analyses can be directly performed on the agarose beads adding lysis or nucleic-acid extraction buffers, or gently eluted with imidazole or EDTA, rendering a fully competent EV preparation. Discussion: This new isolation methodology is based on the recognition of general membrane characteristics independent of surface markers. It is thus unbiased and can be used in any species EV sample, even in samples from animal or plant species against which no suitable antibodies exist. Being an affinity method, the sample handling protocol is very simple, less time-consuming, does not require specialized equipment and can be easily introduced in a clinical automated routine. We demonstrated the high purity and yield of the method in comparison with other commercially available kits. This method can also be scale up or down, with the possibility of analyzing very low amounts of sample, and it is compatible with any downstream analyses thanks to the gentle elution procedure.
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Small extracellular vesicles (sEVs) present fairly distinctive lipid membrane features in the extracellular environment. These include high curvature, lipid-packing defects and a relative abundance in lipids such as phosphatidylserine and ceramide. sEV membrane could be then considered as a "universal" marker, alternative or complementary to traditional, characteristic, surface-associated proteins. Here, we introduce the use of membrane-sensing peptides as new, highly efficient ligands to directly integrate sEV capturing and analysis on a microarray platform. Samples were analysed by label-free, single-particle counting and sizing, and by fluorescence co-localisation immune staining with fluorescent anti-CD9/anti-CD63/anti-CD81 antibodies. Peptides performed as selective yet general sEV baits and showed a binding capacity higher than anti-tetraspanins antibodies. Insights into surface chemistry for optimal peptide performances are also discussed, as capturing efficiency is strictly bound to probes surface orientation effects. We anticipate that this new class of ligands, also due to the versatility and limited costs of synthetic peptides, may greatly enrich the molecular toolbox for EV analysis.
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Biosensing analytical platforms rely on the intimate structure-function relationship of immobilized probes. In this context, hydrogels are appealing semi-wet systems to locally confine biomolecules while preserving their structural integrity and function. Yet, limitations imposed by biomolecule diffusion rates or fabrication difficulties still hamper their broad application. Here, using a self-assembling peptide, a printable and self-adhesive hydrogel was obtained and applied to fabricate arrays of localized bio-functional 3D microenvironments on analytical interfaces. This soft matrix represents a robust and versatile material, allowing fast and selective tuning of analyte diffusion, which is exploited here to run in-gel immunoassays under solution-like conditions in an unprecedented (<10 min) time frame. The developed material overcomes major limitations associated with hydrogels for bioassays, widening the prospects for easy fabrication of multifunctional bio-interfaces for high-throughput, molecular recognition assays.
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Due to significant advances in computational biology, protein prediction, together with antigen and epitope design, have rapidly moved from conventional methods, based on experimental approaches, to in silico-based bioinformatics methods. In this context, we report a reverse vaccinology study that identified a panel of 104 candidate antigens from the Gram-negative bacterial pathogen Burkholderia pseudomallei, which is responsible for the disease melioidosis. B. pseudomallei can cause fatal sepsis in endemic populations in the tropical regions of the world and treatment with antibiotics is mostly ineffective. With the aim of identifying potential vaccine candidates, we report the experimental validation of predicted antigen and type I fimbrial subunit, BPSL1626, which we show is able to recognize and bind human antibodies from the sera of Burkholderia infected patients and to stimulate T-lymphocytes in vitro. The prerequisite for a melioidosis vaccine, in fact, is that both antibody- and cell-mediated immune responses must be triggered. In order to reveal potential antigenic regions of the protein that may aid immunogen re-design, we also report the crystal structure of BPSL1626 at 1.9 Å resolution on which structure-based epitope predictions were based. Overall, our data suggest that BPSL1626 and three epitope regions here-identified can represent viable candidates as potential antigenic molecules.
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Antigen immunoreactivity is often determined by surface regions defined by the 3D juxtapositions of amino acids stretches that are not continuous in the linear sequence. As such, mimicking an antigen immunoreactivity by means of putative linear peptide epitopes for diagnostic purposes is not trivial. Here we present a straightforward and robust method to extend the reach of immune-diagnostic probes design by copresenting peptides belonging to the same antigenic surface. In this case study focused on a computationally predicted Zika virus NS1 protein putative antigenic region, we reached a diagnostic confidence by the oriented and spatially controlled coimmobilization of peptide sequences found adjacent within the protein fold, that cooperatively interacted to provide enhanced immunoreactivity with respect to single linear epitopes. Through our method, we were able to differentiate Zika infected individuals from healthy controls. Remarkably, our strategy fits well with the requirements to build high-throughput screening platforms of linear and mixed peptide libraries, and it could possibly facilitate the rapid identification of conformational immunoreactive regions.
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Anticuerpos/inmunología , Epítopos/inmunología , Péptidos/inmunología , Pruebas Serológicas/métodos , Secuencia de Aminoácidos , Mapeo Epitopo/métodos , Epítopos/química , Humanos , Modelos Moleculares , Sondas Moleculares , Péptidos/química , Conformación Proteica , Curva ROC , Reproducibilidad de los Resultados , Pruebas Serológicas/normas , Relación Estructura-Actividad , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/inmunología , Virus Zika/inmunología , Infección por el Virus Zika/diagnóstico , Infección por el Virus Zika/inmunología , Infección por el Virus Zika/virologíaRESUMEN
Structure-based epitope prediction drives the design of diagnostic peptidic probes to reveal specific antibodies elicited in response to infections. We previously identified a highly immunoreactive epitope from the peptidoglycan-associated lipoprotein (Pal) antigen from Burkholderia pseudomallei, which could also diagnose Burkholderia cepacia infections. Here, considering the high phylogenetic conservation within Burkholderia species, we ask whether cross-reactivity can be reciprocally displayed by the synthetic epitope from B. cenocepacia. We perform comparative analyses of the conformational preferences and diagnostic performances of the corresponding epitopes from the two Burkholderia species when presented in the context of the full-length proteins or as isolated peptides. The effects of conformation on the diagnostic potential and cross-reactivity of Pal peptide epitopes are rationalized on the basis of the 1.8 Å crystal structure of B. cenocepacia Pal and through computational analyses. Our results are discussed in the context of designing new diagnostic molecules for the early detection of infectious diseases.
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Infecciones por Burkholderia/diagnóstico , Burkholderia/inmunología , Mapeo Epitopo/métodos , Epítopos/metabolismo , Inmunoensayo/métodos , Anticuerpos/inmunología , Anticuerpos/fisiología , Proteínas Bacterianas , Infecciones por Burkholderia/microbiología , Clonación Molecular , Simulación por Computador , Regulación Bacteriana de la Expresión Génica , Humanos , Modelos Moleculares , Conformación Proteica , Proteínas RecombinantesRESUMEN
Multiple ligand presentation is a powerful strategy to enhance the affinity of a probe for its corresponding target. A promising application of this concept lies in the analytical field, where surface immobilized probes interact with their corresponding targets in the context of complex biological samples. Here we investigate the effect of multiple epitope presentation (MEP) in the challenging context of IgE-detection in serum samples using peptide microarrays, and evaluate the influence of probes surface density on the assay results. Using the milk allergen alpha-lactalbumin as a model, we have synthesized three immunoreactive epitope sequences in a linear, branched and tandem form and exploited a chemoselective click strategy (CuAAC) for their immobilization on the surface of two biosensors, a microarray and an SPR chip both modified with the same clickable polymeric coating. We first demonstrated that a fine tuning of the surface peptide density plays a crucial role to fully exploit the potential of oriented and multiple peptide display. We then compared the three multiple epitope presentations in a microarray assay using sera samples from milk allergic patients, confirming that a multiple presentation, in particular that of the tandem construct, allows for a more efficient characterization of IgE-binding fingerprints at a statistically significant level. To gain insights on the binding parameters that characterize antibody/epitopes affinity, we selected the most reactive epitope of the series (LAC1) and performed a Surface Plasmon Resonance Imaging (SPRi) analysis comparing different epitope architectures (linear versus branched versus tandem). We demonstrated that the tandem peptide provides an approximately twofold increased binding capacity with respect to the linear and branched peptides, that could be attributed to a lower rate of dissociation (Kd).
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Mapeo Epitopo , Inmunoglobulina E/sangre , Análisis por Matrices de Proteínas , Alérgenos/inmunología , Secuencia de Aminoácidos , Epítopos , Humanos , Hipersensibilidad a la Leche/sangre , PéptidosRESUMEN
BACKGROUND: Identifying physiologically relevant binding partners of amyloid-ß (Aß) that modulate in vivo fibril formation may yield new insights into Alzheimer's disease (AD) etiology. Human cathelicidin peptide, LL-37, is an innate immune effector and modulator, ubiquitous in human tissues and expressed in myriad cell types. OBJECTIVE: We present in vitro experimental evidence and discuss findings supporting a novel hypothesis that LL-37 binds to Aß42 and can modulate Aß fibril formation. METHODS: Specific interactions between LL-37 and Aß (with Aß in different aggregation states, assessed by capillary electrophoresis) were demonstrated by surface plasmon resonance imaging (SPRi). Morphological and structural changes were investigated by transmission electron microscopy (TEM) and circular dichroism (CD) spectroscopy. Neuroinflammatory and cytotoxic effects of LL-37 alone, Aß42 alone, and LL-37/Aß complexes were evaluated in human microglia and neuroblastoma cell lines (SH-SY5Y). RESULTS: SPRi shows binding specificity between LL-37 and Aß, while TEM shows that LL-37 inhibits Aß42 fibril formation, particularly Aß's ability to form long, straight fibrils characteristic of AD. CD reveals that LL-37 prevents Aß42 from adopting its typical ß-type secondary structure. Microglia-mediated toxicities of LL-37 and Aß42 to neurons are greatly attenuated when the two peptides are co-incubated prior to addition. We discuss the complementary biophysical characteristics and AD-related biological activities of these two peptides. CONCLUSION: Based on this body of evidence, we propose that LL-37 and Aß42 may be natural binding partners, which implies that balanced (or unbalanced) spatiotemporal expression of the two peptides could impact AD initiation and progression.
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Péptidos beta-Amiloides/metabolismo , Péptidos beta-Amiloides/farmacología , Amiloide/química , Interleucina-1/farmacología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/farmacología , Amiloide/metabolismo , Amiloide/ultraestructura , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular , Células Cultivadas , Dicroismo Circular , Técnicas de Cocultivo , Humanos , Interleucina-6/metabolismo , Microglía/efectos de los fármacos , Microglía/metabolismo , Microglía/ultraestructura , Microscopía Electrónica de Transmisión , Neuroblastoma/patología , Unión Proteica/efectos de los fármacos , Estructura Secundaria de Proteína , Resonancia por Plasmón de Superficie , Lóbulo Temporal/citología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The interest towards extracellular vesicles (EVs) has grown exponentially over the last few years; being involved in intercellular communication and serving as reservoirs for biomarkers for tumors, they have a great potential for liquid biopsy development, possibly replacing many costly and invasive tissue biopsies. Here we propose, for the first time, the use of a Si/SiO2 interferometric, microarray platform for multiparametric intact EVs analysis combining label-free EVs mass quantitation and high sensitivity fluorescence based phenotyping. Label free interferometric measurement allows to quantify the amount of vesicles captured by printed antibodies while, on the same chip, EVs are also detected by fluorescence in a sandwich immunoassay. The proposed method simultaneously detects, quantify and phenotype intact EVs in a microarray format.
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Vesículas Extracelulares/química , Fluorescencia , Humanos , Espectrometría de Masas , NanopartículasRESUMEN
Efficient diagnosis of emerging and novel bacterial infections is fundamental to guide decisions on therapeutic treatments. Here, we engineered a novel rational strategy to design peptide microarray platforms, which combines structural and genomic analyses to predict the binding interfaces between diverse protein antigens and antibodies against Burkholderia cepacia complex infections present in the sera of Cystic Fibrosis (CF) patients. The predicted binding interfaces on the antigens are synthesized in the form of isolated peptides and chemically optimized for controlled orientation on the surface. Our platform displays multiple Burkholderia-related epitopes and is shown to diagnose infected individuals even in presence of superinfections caused by other prevalent CF pathogens, with limited cost and time requirements. Moreover, our data point out that the specific patterns determined by combined probe responses might provide a characterization of Burkholderia infections even at the subtype level (genomovars). The method is general and immediately applicable to other bacteria.