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1.
Talanta ; 208: 120338, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31816752

RESUMO

Arboviruses have been emerging as a significant global health problem due to the recurrent epidemics. Arboviruses require the development of new diagnostic devices due to the nonspecific clinical manifestations. Herein, we report a biosensor based on cysteine (Cys), zinc oxide nanoparticles (ZnONp), and Concanavalin A (ConA) lectin to differentiate between arboviruses infections. ConA is capable of interacting with the saccharide components of the viral capsid. In this study, we evaluated the reproducibility, sensitivity, and specificity of the sensor for the virus of Dengue type 2 (DENV2), Zika (ZIKV), Chikungunya (CHIKV), and Yellow fever (YFV). Atomic force microscopy measurements confirmed the electrode surface modification and revealed a heterogeneous topography during the biorecognition process. Cyclic voltammetry (CV) and impedance spectroscopy (EIS) were used to characterize the biosensor. The blockage of the oxidation-reduction process is related to the formation of Cys-ZnONp-ConA system on the electroactive area and its subsequent interaction with viral glycoproteins. The sensor exhibited a linear response to different concentrations of the studied arboviruses. Our study demonstrates that ConA lectin recognizes the structural glycoproteins of the DENV2, ZIKV, CHIKV, and YFV. DENV2 is the most structurally similar to ZIKV. Our results have shown that the impedimetric response correlates with the structural glycoproteins, as follow: DENV2 (18.6 kΩ) > ZIKV (14.6 kΩ) > CHIKV (6.86 kΩ) > YFV (5.98 kΩ). The homologous structural regions contribute to ConA-arboviruses recognition. Our results demonstrate the use of the proposed system for the development of biosensors for arboviruses infections.


Assuntos
Infecções por Arbovirus/diagnóstico , Arbovirus/metabolismo , Técnicas Biossensoriais/métodos , Concanavalina A/química , Eletroquímica/métodos , Eletrodos , Nanopartículas Metálicas/química , Infecções por Arbovirus/sangue , Infecções por Arbovirus/virologia , Arbovirus/isolamento & purificação , Febre de Chikungunya/sangue , Febre de Chikungunya/diagnóstico , Febre de Chikungunya/virologia , Vírus Chikungunya/isolamento & purificação , Vírus Chikungunya/metabolismo , Cisteína/química , Dengue/sangue , Dengue/diagnóstico , Dengue/virologia , Vírus da Dengue/isolamento & purificação , Vírus da Dengue/metabolismo , Diagnóstico Diferencial , Glucose/análise , Humanos , Manose/análise , Febre Amarela/sangue , Febre Amarela/diagnóstico , Febre Amarela/virologia , Vírus da Febre Amarela/isolamento & purificação , Vírus da Febre Amarela/metabolismo , Zika virus/isolamento & purificação , Zika virus/metabolismo , Infecção por Zika virus/sangue , Infecção por Zika virus/diagnóstico , Infecção por Zika virus/virologia , Óxido de Zinco/química
2.
Nanoscale ; 11(44): 21155-21166, 2019 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-31663091

RESUMO

Synthetic glycopolymers are increasingly investigated as multivalent ligands for a range of biological and biomedical applications. This study indicates that glycopolymers with a fine-tuned balance between hydrophilic sugar pendant units and relatively hydrophobic polymer backbones can act as single-chain targeted nanocarriers for low molecular weight hydrophobic molecules. Non-covalent complexes formed from poly(triazolyl methacrylate) glycopolymers and low molecular weight hydrophobic guest molecules were characterised through a range of analytical techniques - DLS, SLS, TDA, fluorescence spectroscopy, surface tension analysis - and molecular dynamics (MD) modelling simulations provided further information on the macromolecular characteristics of these single chain complexes. Finally, we show that these nanocarriers can be utilised to deliver a hydrophobic guest molecule, Nile red, to both soluble and surface-immobilised concanavalin A (Con A) and peanut agglutinin (PNA) model lectins with high specificity, showing the potential of non-covalent complexation with specific glycopolymers in targeted guest-molecule delivery.


Assuntos
Portadores de Fármacos/química , Metacrilatos/química , Simulação de Dinâmica Molecular , Polímeros/química , Concanavalina A/química , Aglutinina de Amendoim/química , Espectrometria de Fluorescência
3.
ACS Appl Mater Interfaces ; 11(43): 40944-40950, 2019 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-31597422

RESUMO

An individual nanoparticle-based plasmonic nanotechnology was used for real-time monitoring of lectin-sugar interactions, which could be designed as novel plasmonic nanobiosensors for the detection of trace concanavalin A (ConA) with high sensitivity and selectivity. The localized surface plasmon resonance (LSPR) spectra of Au@Ag nanocubes (NCs) are linearly shifted to a long wavelength with an increasing concentration of ConA. In fact, each Au@Ag NC can act as a nanobiosensor for the quantified detection of trace ConA, which enables the miniaturization of the biosensor system to nanoscale. Furthermore, the results demonstrated the perfect biosensing ability with the dual channel of dark-field microscopy images and LSPR spectra. We expect that this nanobiosensor system can provide an alternative important method for monitoring the specific binding of lectin-sugar at a single nanoparticle surface.


Assuntos
Carboidratos/análise , Concanavalina A/química , Ouro/química , Nanopartículas Metálicas/química , Prata/química , Ressonância de Plasmônio de Superfície
4.
Biomed Res Int ; 2019: 9840890, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31467923

RESUMO

Background: House dust mites are important allergen sources and some of these allergenic proteins may contain carbohydrate moieties, which are able to be isolated using lectins, as Concanavalin A (ConA). This study aimed to investigate allergenicity (IgE) and antigenicity (IgG1 and IgG4) of ConA-unbound and ConA-bound Dermatophagoides pteronyssinus (Dpt) crude extracts using sera of mite-allergic patients as well as inhibition capacity of antibody binding. Material and Methods: We obtained mannose-enriched and mannose-depleted fractions from Dpt by ConA affinity chromatography. Both ConA-bound and ConA-unbound fractions were evaluated by ELISA and Western Blotting for specific IgE, IgG1, and IgG4 reactivity with sera obtained from 95 mite-allergic patients (DP+) and 92 nonallergic (NA) subjects. Inhibition ELISA was used to assess cross-reactivity between Dpt extract and its fractions. Results: Among the DP+ patients, no difference was found between ConA-unbound and ConA-bound fractions regarding the levels of specific IgE, IgG1, and IgG4. Nonallergic subjects had the same levels of specific IgG1 to both ConA-unbound and ConA-bound fractions, although for specific IgG4, values were higher for ConA-bound. A positive correlation was found among specific IgE, IgG1, and IgG4 levels when Dpt was compared to ConA-unbound and ConA-bound fractions. Recognition of crude Dpt by IgE, IgG1, and IgG4 was highly inhibited by ConA-unbound and ConA-bound fractions. Western Blotting revealed a broad spectrum of bands ranging from 14 to 116 kDa recognized by specific IgE and IgG4. However, IgG1 reached higher frequency values on high molecular weight polypeptides. Conclusion: ConA-unbound and ConA-bound fractions derived from D. pteronyssinus crude extract revealed important components involved in the IgE recognition in allergic patients as well as IgG1 and/or IgG4 in allergic and healthy subjects.


Assuntos
Alérgenos/imunologia , Dermatophagoides pteronyssinus/imunologia , Hipersensibilidade/imunologia , Imunoglobulina E/imunologia , Adulto , Alérgenos/química , Animais , Concanavalina A/química , Concanavalina A/imunologia , Dermatophagoides pteronyssinus/química , Ensaio de Imunoadsorção Enzimática , Feminino , Glicosilação , Voluntários Saudáveis , Humanos , Imunoglobulina E/química , Imunoglobulina G/química , Imunoglobulina G/imunologia , Masculino , Pessoa de Meia-Idade , Extratos Vegetais/química , Extratos Vegetais/imunologia , Pyroglyphidae/química , Pyroglyphidae/imunologia
5.
ACS Appl Mater Interfaces ; 11(35): 32366-32372, 2019 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-31397991

RESUMO

Glycopolymers have emerged as powerful and versatile glycan analogues for the investigation of cellular signal transduction. In this study, a layer of the glycopolymer-brush (GlyB) interface was functionalized on the surface of gold substrates. In order to enhance the capability and accessibility of this transducer interface, a combined protocol of copper(0)-mediated living radical polymerization (Cu(0)-LRP) with subsequent "CuAAC" click reaction was utilized to synthesize a set of novel glycopolymer precursors with a tunable scaffold structure and pyranose ligands. The resulting glycopolymer exhibited a fine-tuned molecular weight with a minor dispersity of 1.27. Through surface plasmon resonance (SPR) analysis, various GlyB interfaces exhibiting different saccharide moieties (glucose, mannose, and galactose) were examined to study their adhesion or antiadhesion potential toward three types of proteins, concanavalin A, bovine serum albumin, and peanut agglutinin (PNA). The strong affinity between poly(galactose) and PNA was further employed to construct a proof-of-concept aggregation-mediated sensing system. This minimal naked-eye sensor that consisted of only two substances, namely, gold nanoparticles and glycopolymers, was characterized and tested for its potential in protein quantification.


Assuntos
Concanavalina A/química , Ouro/química , Nanopartículas Metálicas/química , Aglutinina de Amendoim/química , Polissacarídeos/química , Soroalbumina Bovina/química , Ressonância de Plasmônio de Superfície , Animais , Bovinos
6.
Anal Chim Acta ; 1078: 189-199, 2019 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-31358219

RESUMO

Silica-based lectin microcolumns were developed and optimized for the separation and analysis of glycoform fractions in alpha1-acid glycoprotein (AGP) based on both the degree of branching and level of fucosylation. Concanavalin A (Con A) and Aleuria Aurantia lectin (AAL) were immobilized onto HPLC-grade silica by reductive amination and packed into 2.1 mm i.d. × 5.0 cm microcolumns. Factors examined for these microcolumns include their protein content, binding capacity, binding strength and band-broadening under isocratic conditions (Con A) or step elution conditions (AAL) and in the presence of various flow rates or temperatures. These factors were examined by using experiments based on frontal analysis, zonal elution, peak profiling and peak decay analysis. Up to 200 µg AGP could be loaded onto a Con A microcolumn and provide linear elution conditions, and 100 µg AGP could be applied to an AAL microcolumn. The final conditions for separating retained and non-retained AGP glycoform fractions on a Con A microcolumn used a flow rate of 50 µL min-1 and a temperature of 50 °C, which gave a separation of these fractions within 20 min or less. The final conditions for an AAL microcolumn included a flow rate of 0.75 mL min-1, a temperature of 50 °C, and the use of 2.0 mM l-fucose as a competing agent for elution, giving a separation of non-retained and retained AGP glycoforms in 6 min or less. The inter-day precisions were ±0.7-4.0% or less for the retention times of the AGP glycoforms and ±2.2-3.0% or less for their peak areas.


Assuntos
Orosomucoide/análise , Isoformas de Proteínas/análise , Agaricales/química , Cromatografia de Afinidade/instrumentação , Cromatografia de Afinidade/métodos , Concanavalina A/química , Humanos , Proteínas Imobilizadas/química , Lectinas/química , Orosomucoide/química , Isoformas de Proteínas/química , Reprodutibilidade dos Testes , Dióxido de Silício/química
7.
Biosens Bioelectron ; 142: 111506, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31325674

RESUMO

Saccharide sensors represent a broad research area in the scope of sensing devices and their involvement in the medical diagnosis field is particularly relevant for cancer detection at early stage. In that context, we present a non-enzymatic optical fiber-based sensor that makes use of plasmon-assisted tilted fiber Bragg gratings (TFBGs) functionalized for D-glucose biosensing through polydopamine (PDA)-immobilized concanavalin A (Con A). Our probe allows a live and accurate monitoring of the PDA layer deposition leading improved surface biochemistry. The SPR shift observed was assessed to 3.83 ±â€¯0.05 nm within 20 min for a 2 mg/mL dopamine solution. Tests performed in different D-Glucose solutions have revealed a limit of detection close to 10-7 M with the highest sensitivity in the 10-6 to 10-4 M range. This configuration has the capability to overcome the limitations of current enzyme-based solutions.


Assuntos
Concanavalina A/química , Glucose/análise , Indóis/química , Polímeros/química , Ressonância de Plasmônio de Superfície/métodos , Desenho de Equipamento , Humanos , Proteínas Imobilizadas/química , Limite de Detecção , Fibras Ópticas , Ressonância de Plasmônio de Superfície/instrumentação
8.
Biosens Bioelectron ; 139: 111328, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31136921

RESUMO

Quartz crystal microbalance (QCM) methodology has been adopted to unravel important factors contributing to the "cluster glycoside effect" observed in carbohydrate-lectin interactions. Well-defined, glycosylated nanostructures of precise sizes, geometries and functionalization patterns were designed and synthesized, and applied to analysis of the interaction kinetics and thermodynamics with immobilized lectins. The nanostructures were based on Borromean rings, dodecaamine cages, and fullerenes, each of which carrying a defined number of carbohydrate ligands at precise locations. The synthesis of the Borromeates and dodecaamine cages was easily adjustable due to the modular assembly of the structures, resulting in variations in presentation mode. The binding properties of the glycosylated nanoplatforms were evaluated using flow-through QCM technology, as well as hemagglutination inhibition assays, and compared with dodecaglycosylated fullerenes and a monovalent reference. With the QCM setup, the association and dissociation rate constants and the associated equilibrium constants of the interactions could be estimated, and the results used to delineate the multivalency effects of the lectin-nanostructure interactions.


Assuntos
Técnicas Biossensoriais , Lectinas/química , Técnicas de Microbalança de Cristal de Quartzo , Ressonância de Plasmônio de Superfície , Carboidratos/química , Concanavalina A/química , Glicosilação , Ligantes , Nanoestruturas/química , Ligação Proteica
9.
Int J Biol Macromol ; 134: 901-911, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31108148

RESUMO

Lectins are proteins that can bind specifically and reversibly to carbohydrates. This capacity gives lectins multiple biological roles and biotechnological applications. Although lectins can be found in all organisms, plant lectins, especially legume lectins, are undoubtedly the most thoroughly studied. Among legume lectins, the lectin from Canavalia ensiformis (ConA) and Canavalia brasiliensis (ConBr), both from Diocleinae subtribe, are two of the most well-known lectins. It has been 100 years since the first report of ConA and 40 years since the first report of ConBr, making 2019 an important year for lectinology. Structural data of these lectins in combination with biological activity tests clearly indicate that even a small shift in amino acid sequence can affect the tertiary and quaternary structures, consequently affecting the biological activity of these proteins. It is in this context that the present paper aims to review the structural data of ConA and ConBr, focusing on the primary structure, crystallography, tertiary and quaternary structures of these lectins, as well as their binding sites. This paper also expands the structural data by employing molecular dynamics to evaluate carbohydrate-binding properties and structural stability. It is anticipated that these data will increase knowledge about the structure-function relationships of these proteins.


Assuntos
Concanavalina A/química , Lectinas de Plantas/química , Pesquisa , Sequência de Aminoácidos , Sítios de Ligação , Carboidratos/química , Concanavalina A/farmacologia , História do Século XX , História do Século XXI , Modelos Moleculares , Estrutura Molecular , Lectinas de Plantas/farmacologia , Ligação Proteica , Multimerização Proteica , Pesquisa/história , Relação Estrutura-Atividade
10.
Sci Data ; 6(1): 18, 2019 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-30944333

RESUMO

We provide a detailed description of a serial femtosecond crystallography (SFX) dataset collected at the European X-ray free-electron laser facility (EuXFEL). The EuXFEL is the first high repetition rate XFEL delivering MHz X-ray pulse trains at 10 Hz. The short spacing (<1 µs) between pulses requires fast flowing microjets for sample injection and high frame rate detectors. A data set was recorded of a microcrystalline mixture of at least three different jack bean proteins (urease, concanavalin A, concanavalin B). A one megapixel Adaptive Gain Integrating Pixel Detector (AGIPD) was used which has not only a high frame rate but also a large dynamic range. This dataset is publicly available through the Coherent X-ray Imaging Data Bank (CXIDB) as a resource for algorithm development and for data analysis training for prospective XFEL users.


Assuntos
Concanavalina A/química , Proteínas de Plantas/química , Urease/química , Cristalização , Cristalografia por Raios X
11.
J Fluoresc ; 29(3): 609-617, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30963369

RESUMO

This study demonstrates the preparation of photosensitively orientated and crosslinked proteinous polymeric shell having quantum dot based nanocrystals through Amino acid Decorated and Light Underpinning Conjugation Approach (ANADOLUCA). ANADOLUCA is based on photo-electron transfer method and uses these decorated nanocrystals for specifically and effectively recognition and detection of Immunoglobulin M in the aqueous environment. The conjugation method effectively provides an orientation of affinity pairs on the surface of quantum dots nanocrystals. This photosensitive ruthenium-based amino acid monomer is a synthetic and inexpensive material for the preparation of bioconjugates. The nanocrystals give advantages for using a wide pH and temperature range. The construction and preparation method is applicable to silica materials, superparamagnetic particles, quantum dots, carbon nanotubes, Ag/Au nanoparticles, Au surfaces, and polymeric materials. This prepared proteinous polymeric shell decorated nanocrystals are of great potential in applications in life sciences and can be used in infection case studies or allergy symptoms.


Assuntos
Imunoglobulina M/química , Nanopartículas/química , Polímeros/química , Concanavalina A/química , Pontos Quânticos/química , Rutênio/química , Água/química
12.
Macromol Biosci ; 19(6): e1900033, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30977977

RESUMO

Binding of mannose presenting macromolecules to the protein receptor concanavalin A (ConA) is investigated by means of single-molecule atomic force spectroscopy (SMFS) in combination with dynamic light scattering and molecular modeling. Oligomeric (Mw ≈ 1.5-2.5 kDa) and polymeric (Mw ≈ 22-30 kDa) glycomacromolecules with controlled number and positioning of mannose units along the scaffolds accessible by combining solid phase synthesis and thiol-ene coupling are used as model systems to assess the molecular mechanisms that contribute to multivalent ConA-mannose complexes. SMFS measurements show increasing dissociation force from monovalent (≈57 pN) to pentavalent oligomers (≈75 pN) suggesting subsite binding to ConA. Polymeric glycomacromolecules with larger hydrodynamic diameters compared to the binding site spacing of ConA exhibit larger dissociation forces (≈80 pN), indicating simultaneous dissociation from multiple ConA binding sites. Nevertheless, although simultaneous dissociation of multiple ligands could be expected for such multivalent systems, predominantly single dissociation events are observed. This is rationalized by strong coiling of the macromolecules' polyamide backbone due to intramolecular hydrogen bonding hindering unfolding of the coil. Therefore, this study shows that the design of glycopolymers for multivalent receptor binding and clustering must consider 3D structure and intramolecular interactions of the scaffold.


Assuntos
Concanavalina A/química , Substâncias Macromoleculares/química , Manose/química , Receptores de Concanavalina A/química , Concanavalina A/ultraestrutura , Ligação de Hidrogênio , Ligantes , Substâncias Macromoleculares/ultraestrutura , Conformação Molecular , Polímeros/química , Ligação Proteica , Receptores de Concanavalina A/ultraestrutura , Imagem Individual de Molécula , Espectrofotometria Atômica
13.
Acta Crystallogr D Struct Biol ; 75(Pt 2): 192-199, 2019 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-30821707

RESUMO

Harnessing the anomalous signal from macromolecular crystals with volumes of less than 10 000 µm3 for native phasing requires careful experimental planning. The type of anomalous scatterers that are naturally present in the sample, such as sulfur, phosphorus and calcium, will dictate the beam energy required and determine the level of radiation sensitivity, while the crystal size will dictate the beam size and the sample-mounting technique, in turn indicating the specifications of a suitable beamline. On the EMBL beamline P13 at PETRA III, Mesh&Collect data collection from concanavalin A microcrystals with linear dimensions of ∼20 µm or less using an accordingly sized microbeam at a wavelength of 1.892 Š(6.551 keV, close to the Mn edge at 6.549 keV) increases the expected Bijvoet ratio to 2.1% from an expected 0.7% at 12.6 keV (Se K edge), thus allowing experimental phase determination using the anomalous signal from naturally present Mn2+ and Ca2+ ions. Dozens of crystals were harvested and flash-cryocooled in micro-meshes, rapidly screened for diffraction (less than a minute per loop) and then used for serial Mesh&Collect collection of about 298 partial data sets (10° of crystal rotation per sample). The partial data sets were integrated and scaled. A genetic algorithm for combining partial data sets was used to select those to be merged into a single data set. This final data set showed high completeness, high multiplicity and sufficient anomalous signal to locate the anomalous scatterers, and provided phasing information which allowed complete auto-tracing of the polypeptide chain. To allow the complete experiment to run in less than 2 h, a practically acceptable time frame, the diffractometer and detector had to run together with limited manual intervention. The combination of several cutting-edge components allowed accurate anomalous signal to be measured from small crystals.


Assuntos
Canavalia/química , Concanavalina A/química , Cristalização/métodos , Cristalografia por Raios X/métodos , Algoritmos , Cálcio/química , Cátions Bivalentes/química , Cristalização/instrumentação , Cristalografia por Raios X/instrumentação , Coleta de Dados/instrumentação , Coleta de Dados/métodos , Desenho de Equipamento , Manganês/química , Modelos Moleculares , Conformação Proteica , Fluxo de Trabalho
14.
Artif Cells Nanomed Biotechnol ; 47(1): 904-916, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30856018

RESUMO

The aim of present study was to develop the efficient targeting of Concanavalin-A conjugated nanotransfersomal gel to bind directly to melanocytes gel layer against UVB induced skin carcinoma. Carbopol loaded nanotransfersomal gel have prepared by modified rotary evaporation sonication technique & conjugated synthesized by carbodiimide method and they were characterized the morphology, zeta potential, penetration and cell viability. In vitro release studies & skin permeation have determined using Franz diffusion cell and confocal laser scanning microscope (CLSM). The conjugated formulation showed vesicles size, polydispersity index, zeta potential and % conjugation efficiency of 179.0 ± 0.32 nm, 0.197 ± 0.07, 35.1 ± 0.21 mV and 89.73 ± 1.29% respectively. The surface morphology was confirmed by transmission electron microscopy (TEM) and FTIR to make sure the compatibility among its ingredients. Con-A conjugated nanotransfersomal gel showed toxicity on melanoma (A375) in a concentration range of 0.4-2.0 mg/mL, but less toxicity toward HaCaT cells. The MTT assay has analyzed against two different cell lines, to determine their anti-cancer potentials and their targeting ability. Conjugated formulation were found to decrease the cell viability, higher skin targeting efficacy in in-vitro & in-vivo. Concanavalin conjugated nanotransfersomal gel of apigenin promise an efficient and economic approach for the skin cancer.


Assuntos
Apigenina/química , Concanavalina A/química , Sistemas de Liberação de Medicamentos/métodos , Melanoma Experimental/tratamento farmacológico , Preparações Farmacêuticas/administração & dosagem , Neoplasias Cutâneas/tratamento farmacológico , Administração Tópica , Animais , Linhagem Celular Tumoral , Sobrevivência Celular , Liberação Controlada de Fármacos , Estabilidade de Medicamentos , Cabras , Humanos , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos , Tamanho da Partícula , Preparações Farmacêuticas/química , Absorção Cutânea , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Propriedades de Superfície , Raios Ultravioleta
15.
Chem Asian J ; 14(7): 952-957, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30779325

RESUMO

Here we report fluorescence turn-on synthetic lipid rafts by self-assembly of a cationic distyrylanthracene derivative on a negatively-charged sheet in an aqueous solution. First, the negatively-charged 2D membrane structure is formed by lateral associations of aromatic rods with carboxylate groups. Then, the synthetic rafts are floated on the surface of the negatively-charged sheets through electrostatic interactions. The fluorescence of the synthetic rafts is turned on due to the aggregation of the positively-charged AIE dye on the sheets, facilitating monitoring of the formation of rafts. Concanavalin A (Con A) protein can load hierarchically onto the synthetic rafts at neutral pH to provide discrete Con A aggregates with a uniform size of ≈12 nm. The uniform aggregates of Con A on the synthetic rafts can stimulate Jurkat cells with enhanced efficiency, as compared with random-sized aggregates of Con A.


Assuntos
Antracenos/química , Concanavalina A/química , Substâncias Macromoleculares/química , Microdomínios da Membrana/química , Compostos de Amônio Quaternário/química , Estirenos/química , Fluorescência , Humanos , Células Jurkat , Multimerização Proteica/efeitos dos fármacos , Eletricidade Estática
16.
Macromol Biosci ; 19(4): e1800425, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30707496

RESUMO

This study investigates the influence of an increasingly hydrophobic backbone of multivalent glycomimetics based on sequence-defined oligo(amidoamines) on their resulting affinity toward bacterial lectins. Glycomacromolecules are obtained by stepwise assembly of tailor-made building blocks on solid support, using both hydrophobic aliphatic and aromatic building blocks to enable a gradual change in hydrophobicity of the backbone. Their binding behavior toward model lectin Concanavalin A (ConA) is evaluated using isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR) showing higher affinities for glycomacromolecules with higher content of hydrophobic and aromatic moieties in the backbone. Finally, glycomacromolecules are tested in a bacterial adhesion inhibition study against Escherichia coli where more hydrophobic backbones yield higher inhibitory potentials most likely due to additional secondary interactions with hydrophobic regions of the protein receptor as well as a change in conformation exposing carbohydrate ligands for increased binding. Overall, the results highlight the influence and thereby importance of the polymer backbone itself on the resulting properties of polymeric biomimetics.


Assuntos
Aderência Bacteriana , Materiais Revestidos Biocompatíveis/química , Concanavalina A/química , Escherichia coli/metabolismo , Ressonância de Plasmônio de Superfície , Varredura Diferencial de Calorimetria , Escherichia coli/citologia , Interações Hidrofóbicas e Hidrofílicas
17.
Mikrochim Acta ; 186(2): 57, 2019 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-30617909

RESUMO

Disposable syringes were used in a novel point-of-care visual test for detecting pathogenic bacteria (Escherichia coli O157:H7 and Salmonella typhimurium). Hybrid nanoflowers composed of platinum nanoparticles and concanavalin A (Pt-nanoflowers) were prepared through a one-pot reaction and were found to be viable catalase mimics. They catalyze the decomposition of hydrogen peroxide (H2O2) to generate O2. When used as labels in immunoassays, they integrate both the functions of biological recognition and signal amplification. The disposable syringe pressure readout was combined with Pt-nanoflower signal conversion and successfully applied to a visual bacteria detection scheme. Both Escherichia coli O157:H7 and Salmonella typhimurium can be quantified with detection limits of as low as 15 and 7 CFU·mL-1, respectively. Graphical abstract One-pot synthetic platinum nanoparticle (PtNP)-concanavalin A hybrid nanoflowers (Pt-nanoflowers), have been used as ideal signal labels for immunoassays and integrating both essential functions of biological recognition and signal amplification. Disposable syringes were used as a readout to detect pathogenic bacteria.


Assuntos
Escherichia coli O157/isolamento & purificação , Peróxido de Hidrogênio/química , Imunoensaio/métodos , Nanopartículas Metálicas/química , Salmonella typhimurium/isolamento & purificação , Seringas , Animais , Anticorpos/imunologia , Concanavalina A/química , Escherichia coli O157/química , Escherichia coli O157/imunologia , Microbiologia de Alimentos/instrumentação , Microbiologia de Alimentos/métodos , Imunoensaio/instrumentação , Limite de Detecção , Leite/microbiologia , Platina/química , Pressão , Salmonella typhimurium/química , Salmonella typhimurium/imunologia
18.
Biomacromolecules ; 20(2): 969-978, 2019 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-30616349

RESUMO

Biomacromolecules often possess information to self-assemble through low energy competing interactions which can make self-assembly responsive to environmental cues and can also confer dynamic properties. Here, we coupled self-assembling systems to create biofunctional multilayer films that can be cued to disassemble through either molecular or electrical signals. To create functional multilayers, we: (i) electrodeposited the pH-responsive self-assembling aminopolysaccharide chitosan, (ii) allowed the lectin Concanavalin A (ConA) to bind to the chitosan-coated electrode (presumably through electrostatic interactions), (iii) performed layer-by-layer self-assembly by sequential contacting with glycogen and ConA, and (iv) conferred biological (i.e., enzymatic) function by assembling glycoprotein (i.e., enzymes) to the ConA-terminated multilayer. Because the ConA tetramer dissociates at low pH, this multilayer can be triggered to disassemble by acidification. We demonstrate two approaches to induce acidification: (i) glucose oxidase can induce multilayer disassembly in response to molecular cues, and (ii) anodic reactions can induce multilayer disassembly in response to electrical cues.


Assuntos
Substâncias Macromoleculares/química , Quitosana/química , Concanavalina A/química , Eletricidade , Eletrodos , Glucose Oxidase/química , Glicogênio/química , Glicoproteínas/química , Lectinas/química , Eletricidade Estática
19.
Appl Biochem Biotechnol ; 187(1): 1-13, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29869746

RESUMO

Protein glycosylation is one of the most studied post-translational modifications and has received considerable attention for its critical role in the cell biology of eukaryotic cells. The genus Trichoderma has been extensively studied in the biocontrol of soil-borne fungal phytopathogens. The aim of this study was to identify the proteins secreted from Trichoderma harzianum after interacting with the cell walls of two phytopathogens, Sclerotinia sclerotiorum and Fusarium oxysporum. This study used N-glycoprotein enrichment with a concanavalin A (Con A) affinity column, staining detection differential SDS-PAGE, sequencing by mass spectrometric, and protein identification by comparison with the NCBI database to detect the protein expression of the two resulting secretome samples. The majority of the proteins found in both enriched secretomes belonged to a specific class of carbohydrate-active enzymes (CAZymes), within which glycosyl hydrolases (GHs), glycosyltransferases (GTs), and auxiliary activities (AAs) were identified. In this study was described two proteins that have not been previously reported in the secretomes of Trichoderma, a glycosyltransferase (six-harpin) and a galactose oxidase, belonging to the class of auxiliary activities (AA), classified as an AA subfamily AA5-2.The expression pattern of gene encoding to 17 identified proteins, evaluated by real-time quantitative PCR (RT-qPCR), showed significant difference of expression of some GHs and proteases, suggesting a specific gene expression regulation by T. harzianum in presence of different cell walls of two phytopathogens.


Assuntos
Cromatografia de Afinidade/métodos , Concanavalina A/química , Proteínas Fúngicas/metabolismo , Glicoproteínas/metabolismo , Trichoderma/metabolismo , Ascomicetos/metabolismo , Parede Celular/metabolismo , Bases de Dados de Proteínas , Eletroforese em Gel de Poliacrilamida , Proteínas Fúngicas/genética , Fusarium/metabolismo , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Glicoproteínas/genética , Espectrometria de Massas , Reação em Cadeia da Polimerase em Tempo Real , Trichoderma/enzimologia , Trichoderma/genética
20.
Int J Biol Macromol ; 124: 724-732, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30502436

RESUMO

The glucose-responsive behavior of the Con A-sugar affinity based hydrogels, microgels, films and nanoparticles has been widely investigated in the application of drug delivery and biosensors. However, the principle or mechanism of such systems is not well known in literature. In this study, for the first time, the glucose-responsive principle (or principle) of the Con A-sugar based system was detailedly explained through the binding interactions test by isothermal titration calorimetry and ligand binding theory. The study also successfully resolved the controversy regarding the principle of such systems in literature. The other contribution of the study is the experimental validation that the insulin-loaded microgels with different hydrogel-network compositions (based on Con A-sugar affinity) respond to different glucose concentrations. Particularly, the result of the in vitro insulin release suggests that the microgels be able to maintain bolus and basal insulin release in response to different glucose concentrations and the network composition be able to affect the burst release, release rate and overall release amount of insulin. Further, the released insulin has been shown to remain active and the microgels possess no in vitro cytotoxicity to HDF cells. The second contribution is a first step towards the realization of personalized diabetes care.


Assuntos
Concanavalina A/química , Portadores de Fármacos/química , Açúcares/química , Sobrevivência Celular , Dicroísmo Circular , Sistemas de Liberação de Medicamentos , Liberação Controlada de Fármacos , Géis/química , Glucose/metabolismo , Insulina/administração & dosagem , Insulina/farmacocinética , Espectrometria de Fluorescência
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