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1.
Food Chem ; 307: 125514, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31639576

RESUMO

The thermodynamics and kinetics of binding between human serum albumin (HSA) and resveratrol (RES) or its analog (RESAn1) were investigated by surface plasmon resonance (SPR). The binding constant and the kinetic constants of association and dissociation indicated that RESAn1 has higher affinity toward HSA than does RES. The formation of these complexes was entropically driven ( [Formula: see text] , [Formula: see text]  KJ mol-1). However, for both polyphenols, the activation energy (Eact) of association (a) of free molecules was higher than that for dissociation (d) of the stable complex ( [Formula: see text]  KJ mol-1), and the rate of association was faster than that of dissociation since the activation Gibbs free energy (ΔG‡) was lower for the former (ΔGaHSA-RES‡â‰…54.73,ΔGdHSA-RES‡â‰…73.83,ΔGaHSA-RESAn1‡â‰…54.14,ΔGdHSA-RESAn1‡â‰…73.97 KJ mol-1). This study showed that small differences in the structure of polyphenols such as RES and RESAn1 influenced the thermodynamics and kinetics of the complex formation with HSA.


Assuntos
Fenóis/química , Resveratrol/metabolismo , Albumina Sérica Humana/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Proteínas Imobilizadas/química , Proteínas Imobilizadas/metabolismo , Cinética , Ligação Proteica , Resveratrol/química , Albumina Sérica Humana/química , Ressonância de Plasmônio de Superfície , Temperatura Ambiente , Termodinâmica
2.
Analyst ; 144(18): 5353-5367, 2019 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-31384857

RESUMO

Although the traditional strategy of developing general medical treatments for heterogeneous patient populations has a well-established track record, the acknowledgment that one-size-does-not-fit-all is pushing health-care to enter a new era of tailored interventions. The advent of precision medicine is fueled by the high-throughput analysis of individual DNA variants and mRNA expression profiles. However, due to the role of proteins in providing a more direct view of disease states than genomics alone, the ability to comprehensively analyze protein alterations and post translational modifications (PTMs) is a necessary step to unravel disease mechanisms, develop novel biomarkers and targeted therapies. Protein and peptide microarrays can play a major role in this frame, due to high-throughput, low sample consumption and wide applicability. Here, their current role and potentialities are discussed through the review of some promising applications in the fields of PTMs analysis, enzyme screening, high-content immune-profiling and the phenotyping of extracellular vesicles.


Assuntos
Peptídeos/metabolismo , Medicina de Precisão/métodos , Análise Serial de Proteínas/métodos , Bioensaio , Humanos , Proteínas Imobilizadas/química , Proteínas Imobilizadas/metabolismo , Peptídeos/química
3.
Nanoscale ; 11(32): 15139-15146, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31372623

RESUMO

Plasmonic coupling of metallic nanoparticles and adjacent pigments can dramatically increase the brightness of the pigments due to the enhanced local electric field. Here, we demonstrate that the fluorescence brightness of a single plant light-harvesting complex (LHCII) can be significantly enhanced when coupled to a gold nanorod (AuNR). The AuNRs utilized in this study were prepared via chemical reactions, and the hybrid system was constructed using a simple and economical spin-assisted layer-by-layer technique. Enhancement of fluorescence brightness of up to 240-fold was observed, accompanied by a 109-fold decrease in the average (amplitude-weighted) fluorescence lifetime from approximately 3.5 ns down to 32 ps, corresponding to an excitation enhancement of 63-fold and emission enhancement of up to 3.8-fold. This large enhancement is due to the strong spectral overlap of the longitudinal localized surface plasmon resonance of the utilized AuNRs and the absorption or emission bands of LHCII. This study provides an inexpensive strategy to explore the fluorescence dynamics of weakly emitting photosynthetic light-harvesting complexes at the single molecule level.


Assuntos
Complexos de Proteínas Captadores de Luz/química , Proteínas de Plantas/química , Plantas/metabolismo , Ouro/química , Proteínas Imobilizadas/química , Proteínas Imobilizadas/metabolismo , Complexos de Proteínas Captadores de Luz/metabolismo , Microscopia Eletrônica de Transmissão , Nanotubos/química , Proteínas de Plantas/metabolismo , Espectrofotometria , Ressonância de Plasmônio de Superfície
4.
J Chromatogr A ; 1604: 460475, 2019 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-31466701

RESUMO

Enrichment, separation and purification are very important to accurately analyze mycotoxins in complicated samples. In the work, we developed a new enrichment, purification and high-performance liquid chromatography combined with fluorescence detector (HPLC-FLD) for aflatoxins B1 (AFB1), ochratoxin A (OTA) and Zearalenone (ZEN) assay using the macroporous magnetic 3D photonic crystal microspheres (3DPCMs). The conditions of enrichment and purification for mycotoxins have been optimized, which are as follows: pore size of 3DPCMs at 280 nm, 1:1 methanol:acetonitrile (v/v) as eluent, antibody concentrations at 60 µg/mL,60 µg/mL and 120 µg/mL for OTA, AFB1 and ZEN, respectively. The recovery rates in the rice, wheat and corn samples range from 70.01% to 100.12% and the relative standard deviation (RSD) range from 0.45% to 7.09%. The recovery rates used 3DPCMs are almost tenfold higher than that used non-macroporous PCMs in the same conditions. The developed method is simple, rapid (time including enrichment, purification and detection <2 h) and only requires small volume reagents (≤200 µL).


Assuntos
Cromatografia Líquida de Alta Pressão/instrumentação , Cromatografia Líquida de Alta Pressão/métodos , Microesferas , Micotoxinas/análise , Fótons , Aflatoxina B1/análise , Aflatoxina B1/isolamento & purificação , Anticorpos/química , Cristalização , Fluorescência , Proteínas Imobilizadas/química , Micotoxinas/isolamento & purificação , Ocratoxinas/análise , Ocratoxinas/isolamento & purificação , Porosidade , Propriedades de Superfície , Zearalenona/análise , Zearalenona/isolamento & purificação
5.
Molecules ; 24(15)2019 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-31366154

RESUMO

The immobilization of fluorescent proteins is a key technology enabling to fabricate a new generation of photoactive materials with potential technological applications. Herein we have exploited superfolder green (sGFP) and red (RFP) fluorescent proteins expressed with different polypeptide tags. We fused these fluorescent proteins to His-tags to immobilize them on graphene 3D hydrogels, and Cys-tags to immobilize them on porous microparticles activated with either epoxy or disulfide groups and with Lys-tags to immobilize them on upconverting nanoparticles functionalized with carboxylic groups. Genetically programming sGFP and RFP with Cys-tag and His-tag, respectively, allowed tuning the protein spatial organization either across the porous structure of two microbeads with different functional groups (agarose-based materials activated with metal chelates and epoxy-methacrylate materials) or across the surface of a single microbead functionalized with both metal-chelates and disulfide groups. By using different polypeptide tags, we can control the attachment chemistry but also the localization of the fluorescent proteins across the material surfaces. The resulting photoactive material formed by His-RFP immobilized on graphene hydrogels has been tested as pH indicator to measure pH changes in the alkaline region, although the immobilized fluorescent protein exhibited a narrower dynamic range to measure pH than the soluble fluorescent protein. Likewise, the immobilization of Lys-sGFP on alginate-coated upconverting nanoparticles enabled the infrared excitation of the fluorescent protein to be used as a green light emitter. These novel photoactive biomaterials open new avenues for innovative technological developments towards the fabrication of biosensors and photonic devices.


Assuntos
Grafite/química , Proteínas de Fluorescência Verde/química , Hidrogéis/química , Proteínas Imobilizadas/química , Proteínas Luminescentes/química , Proteínas Recombinantes de Fusão/química , Alginatos/química , Técnicas Biossensoriais , Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Histidina/química , Histidina/genética , Histidina/metabolismo , Concentração de Íons de Hidrogênio , Proteínas Imobilizadas/genética , Proteínas Imobilizadas/metabolismo , Luz , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Metacrilatos/química , Nanopartículas/química , Oligopeptídeos/química , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Processos Fotoquímicos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Sefarose/química
6.
Biosens Bioelectron ; 143: 111607, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31445384

RESUMO

As a well-known allergenic indicator in kidney beans, lectins have always been the serious threats for human health. Herein, we introduced a new label-free voltammetric immunosensor for the direct determination of kidney bean lectin (KBL) with potential allergenic activity. Gold nanoparticles-polyethyleneimine-multiwalled carbon nanotubes nanocomposite was one-pot synthesized and modified onto the glass carbon electrode to enhance catalytic currents of oxygen reduction reaction. The KBL polyclonal antibody, acquired from rabbit immunization, was orientedly immobilized on the electrode modified with recombinant staphylococcal protein A via fragment crystallizable (Fc) region of antibody. Under the optimized condition, the immunosensor displayed a good linear response (R2 = 0.978) to KBL with a range from 0.05 to 100 µg/mL and a detection limit of 0.023 µg/mL. Simultaneously, the immunosensor exhibited well selectivity, interference-resistant ability, stability (4 °C) and reproducibility. Compared with the conventional enzyme-linked immunosorbent assay (ELISA) method, the immunosensor was successfully applied to quantify allergenic activity of lectin in raw and cooked (boiled for 30 min) kidney bean milk samples. This new approach provides new perspectives both for rapid quantification of lectin in kidney beans-derived foodstuffs and as a real-time monitoring tool for the allergenic potential during the whole production and consumption process.


Assuntos
Alérgenos/isolamento & purificação , Técnicas Biossensoriais , Lectinas/isolamento & purificação , Alérgenos/imunologia , Animais , Ouro/química , Humanos , Proteínas Imobilizadas/química , Proteínas Imobilizadas/imunologia , Lectinas/imunologia , Nanopartículas Metálicas/química , Nanotubos de Carbono/química , Coelhos
7.
Biosens Bioelectron ; 142: 111512, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31336225

RESUMO

We demonstrate a bionanoelectronic platform for a supported lipid bilayer formed on an MoS2 film for biosensing, biomolecule recognition, and bioelectronic applications. A large-area MoS2 film was synthesized on a sapphire substrate and treated with O2 plasma or Al2O3 deposition to change the surface from hydrophobic to hydrophilic. Measurements of fluorescence and fluorescence recovery after photobleaching confirmed the physical properties of the lipid bilayer on the treated surfaces. We fabricated an electronic device using the treated MoS2 film and characterized the influence of the lipid bilayer on its electrical properties. Furthermore, transmembrane ion channels peptide (gramicidin A) were incorporated into the lipid bilayer and modulations of the electrical properties of the device under various pH conditions and calcium ion were observed. This sensitive and stable platform has strong potential for housing artificial channels and transmembrane ion channels for advanced bioapplications.


Assuntos
Técnicas Biossensoriais/instrumentação , Dissulfetos/química , Bicamadas Lipídicas/química , Molibdênio/química , Nanoestruturas/química , Transistores Eletrônicos , Desenho de Equipamento , Gramicidina/química , Proteínas Imobilizadas/química
8.
Carbohydr Polym ; 222: 115012, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31320069

RESUMO

It is important to control immediate hemorrhage and prevent infection simultaneously in the wound management. However, most of hemostatic materials are associated with low efficiency of hemostasis, poor biocompatibility and lack of antimicrobial properties. A kind of starch-based macroporous sponges (KR-Sps) immobilized covalently with antimicrobial peptide KR12 using highly efficient thiol-ene photo click reaction were developed. The physical properties of these sponges could be fine-tuned by varying the ratio of modified starch/HS-PEG-SH and the polymer concentration. The in vitro and vivo results demonstrated that KR-Sps induced thrombosis, shortened clotting time and reduced the blood loss at bleeding site. Besides, KR12 immobilized sponge exhibited inherent antimicrobial properties against Gram (+) and (-) bacteria and methicillin-resistant Staphylococcus aureus (MRSA), which could maintain at least 5 days. Therefore, KR-Sps were believed to be an excellent candidate as hemostatic and antimicrobial product for the intraoperative wound management.


Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Hemostáticos/farmacologia , Proteínas Imobilizadas/farmacologia , Amido/química , Tampões de Gaze Cirúrgicos , Animais , Antibacterianos/química , Antibacterianos/toxicidade , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/toxicidade , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Materiais Biocompatíveis/toxicidade , Escherichia coli/efeitos dos fármacos , Hemostáticos/química , Hemostáticos/toxicidade , Proteínas Imobilizadas/química , Proteínas Imobilizadas/toxicidade , Masculino , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Camundongos , Testes de Sensibilidade Microbiana , Polietilenoglicóis/química , Polietilenoglicóis/toxicidade , Porosidade , Ratos Sprague-Dawley , Solanum tuberosum/química , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus epidermidis/efeitos dos fármacos , Amido/toxicidade
9.
Carbohydr Polym ; 222: 115021, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31320086

RESUMO

We reported the preparation of antibacterial corn starch film (57% reduction in bacterial count) with enhanced tensile strength (69%) by incorporating immobilized bacteriocin. Whisker shaped crystalline nanocellulose (CNC, length 71.2 ± 20.7 nm and width 27.8 ± 11.2 nm) was prepared from cotton linters by bio-mechanical process, having the degree of polymerization 250. Bacteriocins extracted from broth cultures of P. acidilactici and E. faecium were immobilized on the surface of CNC and used to reinforce the starch film. The biodegradability of reinforced films was affected due to the use of bacteriocin in fillers. Surface morphology and roughness of reinforced films were studied by SEM and AFM. In an ambient environment, the films incorporated with bacteriocin immobilized CNC stayed fresh for 28 days while that of bacteriocin alone had fungal degradation in 14 days. This supports the requirement of CNC immobilization for better stability of bacteriocin on storage.


Assuntos
Antibacterianos/farmacologia , Bacteriocinas/farmacologia , Plásticos Biodegradáveis/química , Celulose/química , Nanopartículas/química , Amido/química , Antibacterianos/química , Bacteriocinas/química , Escherichia coli/efeitos dos fármacos , Proteínas Imobilizadas/química , Proteínas Imobilizadas/farmacologia , Permeabilidade , Solubilidade , Staphylococcus aureus/efeitos dos fármacos , Propriedades de Superfície , Resistência à Tração , Água/química , Zea mays/química
10.
Nature ; 571(7764): 251-256, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31292559

RESUMO

The ability of proteins and other macromolecules to interact with inorganic surfaces is essential to biological function. The proteins involved in these interactions are highly charged and often rich in carboxylic acid side chains1-5, but the structures of most protein-inorganic interfaces are unknown. We explored the possibility of systematically designing structured protein-mineral interfaces, guided by the example of ice-binding proteins, which present arrays of threonine residues (matched to the ice lattice) that order clathrate waters into an ice-like structure6. Here we design proteins displaying arrays of up to 54 carboxylate residues geometrically matched to the potassium ion (K+) sublattice on muscovite mica (001). At low K+ concentration, individual molecules bind independently to mica in the designed orientations, whereas at high K+ concentration, the designs form two-dimensional liquid-crystal phases, which accentuate the inherent structural bias in the muscovite lattice to produce protein arrays ordered over tens of millimetres. Incorporation of designed protein-protein interactions preserving the match between the proteins and the K+ lattice led to extended self-assembled structures on mica: designed end-to-end interactions produced micrometre-long single-protein-diameter wires and a designed trimeric interface yielded extensive honeycomb arrays. The nearest-neighbour distances in these hexagonal arrays could be set digitally between 7.5 and 15.9 nanometres with 2.1-nanometre selectivity by changing the number of repeat units in the monomer. These results demonstrate that protein-inorganic lattice interactions can be systematically programmed and set the stage for designing protein-inorganic hybrid materials.


Assuntos
Silicatos de Alumínio/química , Proteínas Imobilizadas/química , Biossíntese de Proteínas , Nanofios/química , Ligação Proteica
11.
Dalton Trans ; 48(30): 11308-11316, 2019 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-31271177

RESUMO

Complex hierarchical structures are closely associated with their performance in catalysts and protein adsorbents. However, it still remains a great challenge to develop a facile strategy to engineer their structural traits. Herein, we describe a facile strategy combining a hydrothermal reaction and mussel chemistry with a subsequent thermal treatment process for the controllable synthesis of three dimensional hierarchical nickel based composites, which are constructed from MnO nanowire (NW) cores and thin Al2O3@C shells anchored with ultrasmall metallic Ni nanoparticles (NPs). During the processing, MnO2 NWs were utilized as templates for the cores, while the two dimensional NiAl nanosheets were directly adopted as the shell to form three dimensional hierarchical MnO2@NiAl nanowires. After coating with polydopamine-Ni2+ (PDA-Ni2+) and subsequent carbonization under a nitrogen atmosphere, high coverage of metallic Ni NPs and the transformation from MnO2 to MnO cores were all observed in the final product. The size of outer Ni NPs and the morphology of the carbonized product can be tailored by varying the temperature of carbonization, which is also in close association with the performance of catalysis and protein adsorption. Notably, the N-doped carbon layer from polydopamine can act as an electron conductor and facilitate the prevention of the migration and aggregation of the Ni nanoparticles, while the ultrafine Ni nanoparticles can achieve maximum material utilization for catalysis and protein adsorption. In addition, the unique structures can expose more active catalytic or adsorption sites while enabling free diffusion of mass/electron transfer. As a result, the MnO@Al2O3@C/Ni composite exhibited excellent performance in catalysis and protein adsorption.


Assuntos
Proteínas Imobilizadas/química , Nanopartículas Metálicas/química , Nanoestruturas/química , Níquel/química , Proteínas/química , Adsorção , Óxido de Alumínio/química , Carbono/química , Catálise , Compostos de Manganês/química , Óxidos/química
12.
Biosens Bioelectron ; 141: 111469, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31260905

RESUMO

We report on a novel solution immersed silicon (SIS) sensor modified with bio-receptor to detect toluene. To perform this approach, bio-receptor PAS1 which specifically interacts with toluene was chosen as a capture agent for SIS ellipsometric sensing. We constructed wild PAS1 and mutant PAS1 (F46A and F79Y) which are toluene binding-defective. Especially, we utilized an easily accessible capturing approach based on silica binding peptide (SBP) for direct immobilization of PAS1 on the SiO2 surfaces. After the immobilization of SBP-tagged PAS1 to the sensing layers, PAS1-based SIS sensor was evaluated for its ability to recognize toluene. As a result, a significant up-shift in Psi (Ψ) was clearly observed with a low limit of detection (LOD) of 0.1 µM, when treated with toluene on wild PAS1-surface, but not on mutant PAS1-sensing layers, indicating the selective interactions between PAS1 and toluene molecule. The PAS1-SIS sensor showed no changes in Psi (Ψ), if any, negligible, when exposed to benzene, phenol, xylene and 4-nitrophenol as negative controls, thereby demonstrating the specificity of interaction between PAS1 and toluene. Taken together, our results strongly indicate that PAS1-modified ellipsometry sensor can provide a high fidelity system for the accurate and selective detection of toluene.


Assuntos
Proteínas de Bactérias/química , Técnicas Biossensoriais/métodos , Proteínas Quinases/química , Pseudomonas putida/química , Silício/química , Tolueno/análise , Proteínas Imobilizadas/química , Limite de Detecção , Domínios Proteicos , Proteínas Recombinantes de Fusão/química , Poluentes Químicos da Água/análise
13.
Int J Mol Sci ; 20(13)2019 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-31261983

RESUMO

Mesoporous silica are inorganic materials, which are formed by the condensation of sodium silicate or silicon alkoxides around an ordered surfactant used as template [...].


Assuntos
Dióxido de Silício/química , Proteínas Imobilizadas/química , Porosidade , Dióxido de Silício/síntese química
14.
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
15.
Talanta ; 204: 542-547, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31357331

RESUMO

The repeatable immobilization of molecular recognition elements onto particle surfaces has a strong impact on the outcomes of affinity-based assays. In this work, an automatic method for the immobilization of immunoglobulin G (IgG) onto protein A-Sepharose microbeads was established through the flow programming features of the portable lab-on-valve platform using micro-bead injection spectroscopy. The reproducible packing of protein A-microbeads between two optic fibers was feasible, allowing on-column probing of IgG retention. The automation of solutions handling and the precise control of time of IgG interaction with the beads rendered repeatable immobilization cycles, within a short timeframe (<2 min). The proposed method featured the preparation of disposable immunosorbents for downstream analytical applications, such as immunosensing or microenrichment of target analytes. In-situ quantification of IgG@protein A-microbeads was carried out using a horseradish peroxidase-labeled detection IgG. The colorimetric oxidation of 3,3',5,5'-tetramethylbenzidine was monitored on-column. Quantitation of mouse and human IgG immobilized@protein A-microbeads was achieved for loading masses between 0.1 and 0.4 µg per ca. 5.5 mg of sorbent. The implemented detection strategy allowed the quantification of human IgG in certified human serum (ERM®- DA470k/IFCC) and spiked saliva, yielding recoveries of 102-108% and requiring minimal volume (1-15 µL) from serum and saliva.


Assuntos
Cromatografia de Afinidade/métodos , Proteínas Imobilizadas/química , Imunoglobulina G/sangue , Sefarose/análogos & derivados , Proteína Estafilocócica A/química , Animais , Armoracia/enzimologia , Benzidinas/química , Compostos Cromogênicos/química , Colorimetria/métodos , Peroxidase do Rábano Silvestre/química , Humanos , Camundongos , Microesferas , Oxirredução , Saliva/química , Sefarose/química
16.
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
17.
Biomater Sci ; 7(8): 3204-3212, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31147655

RESUMO

The intrinsically disordered Parkinson disease protein α-synuclein (αS) performs conformational changes induced by intermolecular protein-protein as well as by protein-membrane interactions. Aggregation of αS is a hallmark for the disease, however the role of the membrane in the aggregation process still needs to be clarified. We used a surface-enhanced infrared absorption (SEIRA) spectroscopic approach to investigate the effect of lipid interactions on αS conformation. The near-field detection of SEIRA allows to study exclusively structural changes of immobilized αS with the advantage that the supernatant remains undetected and thus does not interfere with the spectral read-out. self-assembled monolayer (SAMs) of mixed NHS-PEG-SH linker and MT(PEG)4 spacer molecules were utilized to immobilize αS. The linker/spacer composition of the SAM was adjusted to prevent αS-αS interactions. Two different methods were applied for site-specific (C-terminal and N-terminal) αS immobilization. The immobilized protein was then exposed to lipid vesicles and SEIRA difference spectra were recorded to monitor the αS conformation over time. Irrespective of the used immobilization method, αS tethering hindered lipid-induced conformational changes. The spectra also indicate that a fraction of the immobilized αS eventually desorbs from the surface into the supernatant solution. Desorbed αS performs conformational changes and formation of ß-structured aggregates is observed upon interaction with either lipid vesicles or supplementary αS. Our study demonstrates that αS aggregates only when the protein is free in solution and that surface immobilization procedures, commonly used in many analytical applications, can change the dynamic behavior of proteins thereby affecting protein structure and function.


Assuntos
Proteínas Imobilizadas/química , Proteínas Imobilizadas/metabolismo , Lipídeos de Membrana/metabolismo , Espectrofotometria Infravermelho , alfa-Sinucleína/química , alfa-Sinucleína/metabolismo , Ouro/química , Fosfatidilgliceróis/metabolismo , Polietilenoglicóis/química , Ligação Proteica , Propriedades de Superfície
18.
Mikrochim Acta ; 186(7): 411, 2019 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-31183566

RESUMO

This paper describes a dual electrochemical immunoassay for the simultaneous determination of IL-13Rα2 and CDH-17, two biomarkers of emerging relevance in metastatic processes. The sandwich assay uses a screen-printed dual carbon electrode that was electrochemically grafted with p-aminobenzoic acid to allow the covalent immobilization of capture antibodies. A hybrid composed of graphene quantum dots (GQDs) and multiwalled carbon nanotubes (MWCNTs) act as nanocarriers for the detection antibodies and horseradish peroxidase. The use of this hybrid material considerably improves the assay (in comparison to the use of MWCNTs) due to the peroxidase mimicking activity of the GQDs. The method works at a low working potential (0.20 V vs. Ag pseudo-reference electrode) and thus is not readily interfered by unknown electroactive species. The dual immunoassay allows for the selective determination of both biomarkers with LOD values of 1.4 (IL-13sRα2) and 0.03 ng mL-1 (CDH-17). The simultaneous determination of IL-13Rα2 and CDH-17 was accomplished in lysates from breast and colorectal cancer cells with different metastatic potential, and in paraffin-embedded tumor tissues extracts from patients diagnosed with colorectal cancer at different stages. The applicability to discriminate the metastatic potential even in intact cells through the detection of both extracellular receptors has been demonstrated also. The assay can be performed within 3 h, requires small sample amounts (0.5 µg), and has a simple protocol. Graphical abstract Dual amperometric immunosensing of the metastasis-related biomarkers IL-13Rα2 and CDH-17 in human colorectal cancer cells and tissues by using grafted screen-printed electrodes and composites of quantum dots and carbon nanotubes as nanocarriers.


Assuntos
Biomarcadores Tumorais/análise , Caderinas/análise , Imunoensaio/métodos , Subunidade alfa2 de Receptor de Interleucina-13/análise , Nanotubos de Carbono/química , Pontos Quânticos/química , Técnicas Biossensoriais/métodos , Linhagem Celular Tumoral , Técnicas Eletroquímicas , Eletrodos , Grafite/química , Humanos , Proteínas Imobilizadas/química , Limite de Detecção , Metástase Neoplásica/diagnóstico , Sensibilidade e Especificidade
19.
Int J Biol Macromol ; 136: 106-114, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31185240

RESUMO

Magnetic nanoparticles coated with polymer shell containing reactive functional groups are of great interest especially as substrates for immobilization of ligands in biomedicine and catalysis. This article describes synthesis of novel functional MNPs coated with aminated starch via simple, fast and efficient method of functionalization of the surface by one-minute pounding in mortar. The concept is based on simplifying the synthesis of the magnetic support and obtaining a material that allows for effective bioligand immobilization. Basing on our previous research in the area of MNPs synthesis and biomedical applications, the high yield (149.96 mg/g of support) and effective immobilization of HSA was demonstrated for these nanoparticles without loss of protein activity. Obtained materials were characterized with ATR-FTIR spectroscopy, scanning (SEM) and transmission (TEM) electron microscopy, dynamic light scattering (DLS), X-ray diffraction, TGA-DTA and SQUID analysis. The developed method allows for modification of polysaccharides and nanoparticles towards materials enriched with amino groups in a quick and easy way. It can be expected that this method of quick solvent-free amination will find application in the chemistry of materials and polymers. In addition, the new obtained amino-rich MNPs may find use as carriers for the immobilization of bioligands in catalysis and pharmaceutical analysis.


Assuntos
Proteínas Imobilizadas/química , Nanopartículas de Magnetita/química , Polímeros/química , Albumina Sérica Humana/química , Amido/química , Aminação , Humanos , Proteínas Imobilizadas/metabolismo , Cinética , Polissacarídeos/química , Albumina Sérica Humana/metabolismo , Propriedades de Superfície , Temperatura Ambiente
20.
Biosens Bioelectron ; 141: 111361, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31207570

RESUMO

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) ribonucleoprotein (RNP) complex is an RNA-guided DNA-nuclease that is part of the bacterial adaptive immune system. CRISPR/Cas9 RNP has been adapted for targeted genome editing within cells and whole organisms with new applications vastly outpacing detection and quantification of gene-editing reagents. Detection of the CRISPR/Cas9 RNP within biological samples is critical for assessing gene-editing reagent delivery efficiency, retention, persistence, and distribution within living organisms. Conventional detection methods are effective, yet the expense and lack of scalability for antibody-based affinity reagents limit these techniques for clinical and/or field settings. This necessitates the development of low cost, scalable CRISPR/Cas9 RNP affinity reagents as alternatives or augments to antibodies. Herein, we report the development of the Streptococcus pyogenes anti-CRISPR/Cas9 protein, AcrIIA4, as a novel affinity reagent. An engineered cysteine linker enables covalent immobilization of AcrIIA4 onto glassy carbon electrodes functionalized via aryl diazonium chemistry for detection of CRISPR/Cas9 RNP by electrochemical, fluorescent, and colorimetric methods. Electrochemical measurements achieve a detection of 280 pM RNP in reaction buffer and 8 nM RNP in biologically representative conditions. Our results demonstrate the ability of anti-CRISPR proteins to serve as robust, specific, flexible, and economical recognition elements in biosensing/quantification devices for CRISPR/Cas9 RNP.


Assuntos
Proteínas de Bactérias/análise , Bacteriófagos/química , Técnicas Biossensoriais/métodos , Proteína 9 Associada à CRISPR/análise , Streptococcus pyogenes/química , Proteínas Virais/química , Sistemas CRISPR-Cas , Proteínas Imobilizadas/química , Ligantes , Modelos Moleculares
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